High-complexity synthetic gut bacterial communities

Abstract

To provide high-complexity defined gut microbial communities capable of achieving substantial engraftment and having stability following human fecal community microbial exposure and to provide methods of producing the same.SOLUTION: Also there are provided methods of using high-complexity defined gut microbial communities for the treatment of dysbiosis or a pathological condition in an animal. Disclosed herein is a high-complexity defined gut microbial community comprising a plurality of between 40 and 500 defined microbial strains, the defined gut microbial community achieving substantial engraftment when administered to a gnotobiotic mouse, and the engrafted defined gut microbial community being stable following a human fecal community microbial exposure.SELECTED DRAWING: None

Description

[Technical field]

[0001] STATEMENT REGARDING RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. DK113598 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 770,706, filed November 21, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0003] Fecal microbiota transplantation (FMT) is a promising therapeutic approach that has proven highly effective in treating conditions such as recurrent C. difficile infection (CDI). To avoid the inconvenience of using stool, Allen-Vercoe and Petrof proposed the treatment of recurrent CDI using a synthetic bacterial ecosystem 33 strains developed from a subset of isolates. Allen-Vercoe, E. and Petrof, EO, 2013, "Artificial stool transplantation: progress towards a safer, more effective and acceptable alternative," Expert Rev. Gastroenterol. Hepatol. 7(4), 291-293 (2013); WO 2013 / 037068.

[0004] FMT has been proposed by Fischbach et al. as a therapeutic intervention that alters the diverse metabolites in a patient's bloodstream, urine, bile, and / or stool by manipulating the molecular output of the gut bacterial community. Dodd et al., 2017, "A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites," Nature 551: 648-652;Fischbach MA, 2018, “Microbiome: Focus on Causation and Mechanism,” Cell 174(4):785-790. Although FMT shows great promise as a therapeutic approach, better implantable compositions are needed as well as better methods for developing therapeutic agents with the desired activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 037068 [Non-patent literature]

[0006] [Non-Patent Document 1] Allen-Vercoe, E. and Petrof, EO, 2013, “Artificial stool transplantation: progress towards a safer, more effective and acceptable alternative,” Expert Rev. Gastroenterol. Hepatol. 7(4), 291-293 (2013) [Non-Patent Document 2] Dodd et al., 2017, “A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites,” Nature 551: 648-652 [Non-Patent Document 3] Fischbach MA, 2018, “Microbiome: Focus on Causation and Mechanism,” Cell 174(4):785-790 Summary of the Invention [Means for solving the problem]

[0007] Disclosed herein is a high complexity defined gut microbial community comprising a plurality of between 40-500 defined microbial strains, wherein the defined gut microbial community achieves substantial engraftment when administered to gnotobiotic mice, and the engrafted defined gut microbial community is stable following exposure to human fecal community microbes. In some embodiments, the stability of the high complexity defined gut microbial communities disclosed herein is characterized by up to 10% of the defined microbial strains dropping out following microbial exposure and / or up to 10% of new strain emergence resulting from the human fecal community appearing following microbial exposure.

[0008] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the defined microbial strains of the high complexity defined gut microbial community are detectable following microbial exposure.

[0009] In some embodiments, the stability of the high-complexity defined gut microbial community is characterized by metagenomic analysis of fecal samples obtained from mice after microbial exposure. In some embodiments, the metagenomic analysis is selected from whole genome sequencing, ribosomal gene sequencing, or ribosomal RNA sequencing. In certain embodiments, the metagenomic analysis is whole genome shotgun sequencing.

[0010] In some embodiments, the high complexity defined gut microbial community disclosed herein comprises between 100-200 or between 100-130 microbial strains.

[0011] In some embodiments, each defined microbial strain of the high complexity defined gut microbial community disclosed herein is molecularly identified.In some embodiments, molecular identification comprises the identification of nucleic acid sequences that uniquely identify each defined microbial strain.In certain embodiments, molecular identification comprises the identification of 16S rRNA nucleic acid sequences or whole genome sequencing.In some embodiments, molecular identification comprises matrix-assisted laser desorption / ionization time-of-flight mass spectrometry that uniquely identifies the defined microbial species.

[0012] In some embodiments, the high complexity defined gut microbial communities disclosed herein reduce the number of C. difficile colony forming units (CFU) per μl of feces by at least 1-2 log, at least 2-3 log, at least 3-4 log, at least 4-5 log, or at least 5-6 log when tested in a mouse model of persistent Clostridium difficile infection.

[0013] In some embodiments, the high complexity defined gut microbial community disclosed herein significantly alters the profile and / or concentration of bile acids present in the feces of mice compared to isogenic gnotobiotic control mice. In certain embodiments, the bile acids are selected from the group consisting of Tβ-MCA, Tα-MCA, TUDCA, THDCA, TCA, 7β-CA, 7-oxo-CA, TCDCA, Tω-MCA, TDCA, α-MCA, β-MCA, ω-MCA, Muro-CA, d4-CA, CA, TLCA, UDCA, HDCA, CDCA, DCA and LCA.

[0014] In some embodiments, the high complexity defined gut microbial communities disclosed herein significantly alter the concentration of one or more metabolites in the urine, blood, or feces of mice compared to isogenic gnotobiotic control mice. In certain embodiments, the metabolite is selected from the group consisting of 4-hydroxybenzoic acid, L-tyrosine, 4-hydroxyphenylacetic acid, DL-p-hydroxyphenyllactic acid, p-coumaric acid, 3-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)pyruvic acid, indole-3-carboxylic acid, tyramine, L-phenylalanine, phenylacetic acid, 3-indoleacetic acid, DL-3-phenyllactic acid, L-tryptophan, DL-indole-3-lactic acid, phenylpyruvic acid, trans-3-indoleacrylic acid, 3-indolepyropionic acid, 3-phenylproprionic acid, trans-cinnamic acid, tryptamine, phenol, indole-3-carboxaldehyde, p-cresol, indole, 4-vinylphenol, and 4-ethylphenol.

[0015] In some embodiments, one or more of the defined microbial strains of the high complexity defined gut microbial communities disclosed herein have at least 2, at least 3, at least 5, at least 10, or at least 13 metabolic phenotypes selected from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production.

[0016] In some embodiments, the defined microbial strains of the high complexity defined gut microbial community disclosed herein are selected from the group consisting of Acidaminococcus fermentans DSM 20731, Acidaminococcus sp. D21, Akkermansia muciniphila ATCC BAA-835, Alistipes putredinis DSM 17216, Anaerofustis stercorihominis DSM 17244, Anaerostipes caccae DSM 14662, Anaerotruncus colihominis DSM 17241, Bacteroides caccae ATCC 43185, Bacteroides cellulosilyticus DSM 14838, Bacteroides coprocola DSM 17136, Bacteroides coprophilus DSM 18228, Bacteroides dorei 5_1_36 / D4(HM 29), Bacteroides dorei DSM 17855, Bacteroides eggerthii DSM 20697, Bacteroides finegoldii DSM 17565, Bacteroides fragilis 3_1_12, Bacteroides intestinalis DSM 17393, Bacteroides ovatus ATCC 8483, Bacteroides pectinophilus ATCC 43243, Bacteroides plebeius DSM 17135, Bacteroides sp.1_1_6, Bacteroides sp.2_1_16, Bacteroides sp.2_1_22, Bacteroides sp.3_1_19, Bacteroides sp.9_1_42FAA, Bacteroides sp.D2, Bacteroides stercoris ATCC 43183 DSMZ 19555, Bacteroides thetaiotaomicron VPI-5482, Bacteroides uniformis ATCC 8492, Bacteroides vulgatus ATCC 8482, Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836, Bifidobacterium adolescentis L2-32, Bifidobacterium breve DSM 20213, Bifidobacterium catenulatum DSM 16992, Bifidobacterium longum infantis ATCC 55813, Bifidobacterium pseudocatenulatum DSM 20438, Blautia. hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Bryantella formatexigens DSM 14469、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathawayi DSM 13479、Clostridium hylemonas DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.L2-50、Clostridium sp.M62 / 1、Clostridium spiroforme DSM 1552、Clostridium sporogenes ATCC 15579、Collinsella aerofaciens ATCC 25986、Collinsella dung DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dialister invisus DSM 15470, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Lactobacillus ruminis ATCC 25644, Lactococcus lactis subsp.lactis Il1403 -> sub DSMZ 20729, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Olsenella uli DSM 7084, Parabacteroides distasonis ATCC 8503, Parabacteroides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp.D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii L2-32, Ruminococcus flavefaciens FD 1. Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9(ATCC 19258), Subdoligranulum variabile DSM 15176, Veillonella dispar ATCC 17748, Veillonella sp.3_1_44 HM 64, and Veillonella sp.6_1_27 HM 49.

[0017] In some embodiments, the defined microbial strains of the high complexity defined gut microbial community disclosed herein are selected from the group consisting of Acidaminococcus fermentans DSM 20731, Acidaminococcus sp. D21, Adlercreutzia equolifaciens DSM 19450, Akkermansia muciniphila ATCC BAA-835、Alistipes finegoldii DSM 17242、Alistipes ihumii AP11、Alistipes indistinctus YIT 12060 / DSM 22520、Alistipes onderdonkii DSM 19147、Alistipes putredinis DSM 17216、Alistipes senegalensis JC50 / DSM 25460、Alistipes shahii WAL 8301 / DSM 19121、Anaerofustis stercorihominis DSM 17244、Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides rodentium DSM 26882、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis、ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium breve、Bifidobacterium catenulatum DSM 16992、Bifidobacterium pseudocatenulatum DSM 20438、Bilophila wadsworthia ATCC 49260、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Blautia sp.KLE 1732(HM 1032)、Blautia wexlerae DSM 19850、Bryantella formatexigens DSM 14469、Burkholderiales bacterium 1_1_47、Butyricimonas virosa DSM 23226、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridiales bacterium VE202-03、Clostridiales bacterium VE202-14、Clostridiales bacterium VE202-27、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathewayi DSM 13479、Clostridium hylemonae DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.ATCC 29733 VPI C48-50、Clostridium sp.L2-50、Clostridium sp.M62 / 1, Clostridium spiroforme DSM 1552, Collinsella aerofaciens ATCC 25986, Collinsella stercoris DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC. 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Intestinimonas butyriciproducens DSM 26588, Lactobacillus ruminis ATCC 25644, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Odoribacter splanchnicus DSM 20712, Olsenella uli DSM 7084, Oscillibacter sp.KLE 1728, Parabacteroides distasonis ATCC 8503, Parabacteroides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp.D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii ATCC, Ruminococcus flavefaciens FD 1, Ruminococcus gauvreauii DSM 19829, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum sp. 4_3_54A2FAA, Subdoligranulum variabile DSM 15176, and Veillonella dispar ATCC 17748.

[0018] In some embodiments, the present disclosure provides a method of treating an animal having a microbial incompatibility or condition comprising administering a high complexity defined gut microbial community as disclosed herein. In certain embodiments, the animal is a mammal, more particularly a human.

[0019] In some embodiments, the present disclosure provides a method for treating persistent C.difficile infection by administering a high-complexity defined gut microbial community as disclosed herein.In some embodiments, the present disclosure provides a method for treating cholestatic disease by administering a high-complexity defined gut microbial community as disclosed herein.In certain embodiments, the cholestatic disease is primary sclerosing cholangitis, primary biliary cholangitis, progressive familial intrahepatic cholestasis, or nonalcoholic steatohepatitis.

[0020] In some embodiments, the high complexity defined gut microbial community disclosed herein is administered via a route selected from the group consisting of oral, rectal, fecal (by enema), and naso / oral gavage.

[0021] In some embodiments, each of the multiple defined microbial strains is cultured individually and then combined to form a high complexity defined gut microbial community. In other embodiments, all of the multiple defined microbial strains are cultured together to form a high complexity defined gut microbial community. In further embodiments, one or more of the multiple defined microbial strains are cultured individually, two or more of the defined microbial strains are cultured together, and the individually cultured defined microbial strains and the co-cultured defined microbial strains are combined together to form a defined gut microbial community.

[0022] The present disclosure also provides a formulation comprising the high complexity defined gut microbial community disclosed herein and a pharma- ceutically acceptable carrier or excipient.

[0023] Also provided is a method for generating a high complexity defined gut microbial community by in vivo backfilling, the method comprising: i) combining a plurality of defined microbial strains; ii) engrafting the combined multiple defined microbial strains in the gut of an animal to generate an engrafted animal; iii) exposing the engrafted animals to a human stool sample; iv) maintaining the exposed engrafted animals for a time sufficient for intestinal colonization of the animals with the microbial strains from the human stool sample, thereby generating an intestinal community in the intestine of the animals; v) identifying microbial strains in the gut community by metagenomic analysis; vi) identifying whether there are differences between the microbial strains that make up the gut community and the microbial strains that make up the combined plurality of defined microbial strains; vii) if there is a significant difference between the microbial strains constituting the gut community and the combined plurality of defined microbial strains, adding one or more additional defined microbial strains that were not present in step i) to the combined plurality of defined microbial strains or removing defined microbial strains that were present in the combined plurality of defined microbial strains of step i) to generate a modified combined plurality of defined microbial strains, and repeating steps ii) to vi) in the non-engrafted animals using the modified combined plurality of defined microbial strains as the combined plurality of defined microbial strains. and if minimal differences exist, the modified defined microbial community of the final step vii) is a high complexity defined gut microbial community.

[0024] In certain embodiments, step i) of the method for generating a high complexity defined gut microbial community comprises the steps of: fructan, inulin, glucuronoxylan, arabinoxylan, glucomannan, beta-mannan, dextran, starch, arabinan, xyloglucan, galacturonan, beta-glucan, galactomannan, rhamnogalacturonan I, rhamnogalacturonan II, arabinogalactan, mucin O-linked glycan, yeast alpha-mannan, yeast beta-glucan, chitin, alginate, porphyrin, laminarin, carrageenan, agarose, alternan, levan, xanthan gum, galactooligosaccharides, hyaluronan, chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, phenylalanine, tyrosine, tryptophan, leucine, valine, isoleucine, , glycine, proline, asparagine, glutamine, aspartic acid, glutamic acid, cysteine, lysine, arginine, serine, methionine, alanine, arginine, histidine, ornithine, citrulline, carnitine, hydroxyproline, cholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholesterol, cinnamic acid, coumaric acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, chlorogenic acid, catechin, epicatechin, gallic acid, pyrogallol, catechol, quercetin, myricetin, campherol, luteolin, apigenin, naringenin, and hesperidin. In some embodiments, the combined plurality of defined microbial strains is capable of metabolizing at least 2, at least 4, at least 8, at least 12, at least 24, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or all of the substrates listed above.

[0025] In certain embodiments, a method for generating a high complexity defined gut microbial community comprises: viii) performing a C. difficile plate count on fecal samples obtained from animals with persistent C. difficile infection; ix) engrafting the high complexity defined gut microbial community into the gut of an animal with persistent C. difficile infection to generate an engrafted infected animal; x) maintaining the engrafted infected animals for a time sufficient for intestinal colonization with microbial strains of a high complexity defined gut microbial community, thereby generating an engrafted infected community in the intestine of the engrafted infected animals; xi) performing additional C. difficile plate counts on fecal samples obtained from engrafted infected animals; xii) if the number of C. difficile CFUs obtained from the plate count in step xi) is not significantly less than the number of C. difficile CFUs obtained from the plate count in step viii), adding one or more additional defined microbial strains that were not present in step ix) to the high complexity defined gut microbial community to generate a modified high complexity defined gut microbial community, and repeating steps viii)-xi) in animals with non-engrafted, persistent C. difficile infection using the modified high complexity defined gut microbial community as the high complexity defined gut microbial community. Further comprising: If there is a statistically significant reduction in the CFU count of C. difficile obtained from the plate count in step xi) compared to the CFU count of C. difficile obtained from the plate count in step viii), the modified high complexity defined gut microbial community in final step xi) is the final high complexity defined gut microbial community. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a schematic illustrating the workflow for the preparation of high-complexity defined gut microbial communities.

[0027] [Diagram 2]FIG. 2 is a graph showing the relative abundance of microbial strains in mice colonized with a defined microbial community of high complexity and challenged with stool samples prepared from three different human donors.

[0028] [Diagram 3] Figure 3A is a schematic diagram of the treatment schedule for gnotobiotic mice colonized with human stool samples, inoculated with C. difficile, and treated with a high-complexity defined gut microbial community, and Figure 3B is a dot plot of C. difficile concentrations in feces of mice treated according to the treatment schedule in Figure 3A.

[0029] [Figure 4] FIG. 4 is a bar graph of bile acid concentrations in feces (FIG. 4A) and cecum (FIG. 4B) from mice treated with human fecal samples or high complexity defined gut microbial communities.

[0030] [Diagram 5] FIG. 5 is a bar graph of metabolite concentrations in human fecal samples or urine samples from mice treated with high complexity defined gut microbial communities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] 1.Definition The terms "a" and "an" are used herein to mean "one or more" and include the plural unless the context requires otherwise.

[0032] As used herein, the "abundance" of a particular intestinal microorganism refers to the number of individual organisms in the intestine of an individual. Abundance can be described as a proportion of the total intestinal population (e.g., number of organisms relative to the total intestinal population, amount of organism relative to the mass of the total intestinal population).

[0033] As used herein, "animal" refers to an organism to be treated with a microbial community, e.g., a high complexity defined gut microbial community. Animals include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0034] As used herein, "dysbiosis" refers to the state of the intestinal microbiome of an animal in which normal diversity and / or function is disrupted. In some cases, dysbiosis can result from a decrease in the diversity of the intestinal microbiota, an overabundance of one or more pathogens or pathogenic symbionts, or the presence of pathogenic symbionts.

[0035] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desired results.

[0036] As used herein, "humanized mouse" refers to a mouse with a human gut microbiome. A humanized mouse can be generated by removing the mouse's gut flora (e.g., by administering PEG-3350 and electrolytes, e.g., GoLYTELY® (Braintree Laboratories, Inc., Braintree, MA)) and / or administering broad-spectrum antibiotics, and colonizing the mouse with a preparation of microorganisms from human stool. A humanized mouse can also refer to a gnotobiotic mouse colonized with a human stool sample. In some embodiments, the intestine of the humanized mouse can be flushed (e.g., by administering PEG-3350) and then inoculated with the high-complexity gut microbial community described herein.

[0037] As used herein, "isogenic gnotobiotic control mice" refer to mice used as experimental controls that share the same genotype as the mice receiving a microbial community, e.g., a high complexity defined gut microbial community, but are administered a vehicle control, or other experimental negative control.

[0038] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline (PBS) solutions, water, emulsions (such as oil / water or water / oil emulsions), and various wetting agents. The compositions may also include stabilizers and preservatives. Examples of carriers, stabilizers, and adjuvants are described, for example, in Martin, See Remington's Pharmaceutical Sciences, 15th Ed. Mack Publ. Co., Easton, PA (1975).

[0039] As used herein, the "prevalence" of an intestinal microorganism refers to the frequency with which the organism is found in the human intestine (eg, number of individuals in a population).

[0040] As used herein, "significantly" or "significant" refers to a change or alteration in a measurable parameter to a degree that is statistically significant as determined according to an appropriate statistically relevant test. For example, in some embodiments, a change or alteration is significant if it is statistically significant according to, for example, a Student's t-test, a chi-squared test, or a Mann-Whitney test.

[0041] As used herein, "minimally different" refers to a change or alteration in a measurable parameter that is not statistically significant as determined according to an appropriate statistically relevant test. For example, in some embodiments, a change or alteration is minimally different if it is not statistically significant according to, for example, a Student's t-test, a chi-squared test, or a Mann-Whitney test. 2. Fecal microbiota transplantation

[0042] Fecal microbiota transplantation (FMT) is notable for two things that suggest its generality: 1) the acute adverse event rate is very low, suggesting that the procedure appears to be generally safe, and 2) it has already worked very well to treat certain conditions, even without concerted efforts to optimize the engraftment process. Taken together, these findings counterintuitively suggested to the inventors that a single community could in principle be stably transplanted into the gut of millions of patients, and that administration of a high-complexity defined gut microbial community could be safer and more predictable than ostensibly simple perturbations to the gut (e.g., adding or removing one or a few strains). This is exciting, since administration of a high-complexity defined gut microbial community is the biggest "lever" that can be pulled in terms of controlling human biology related to the microbiota. However, the current prior art, fecal transplantation, cannot be scaled. For this reason, new technologies are needed that allow the design and assembly of transplantable communities that are fully defined on the one hand and address the complexity of the natural gut community on the other. 3. Microbial community

[0043] As used herein, "community" or "microbial community" refers to a physical combination of multiple different microorganisms, usually multiple different bacterial strains, and may include one or more strains or archaea. A naturally occurring gut microbiome is one example of a community. An artificially created mixture of strains with known identity is another example of a community. A defined gut microbial community is yet another example of a community. As used herein, "defined gut microbial community" refers to a combination of multiple microbial strains for engraftment in the intestine of an animal, where each microbial strain is molecularly identified.

[0044] As used herein, "microbial strain" refers to a type or subtype of a microorganism. As used herein, a "defined microbial strain" is a microbial strain that has been molecularly identified, for example, a microbial strain whose entire genome has been sequenced. As used herein, a "multiple defined microbial strains" refers to two or more microbial strains from two or more distinct microbial species. In some embodiments, multiple microbial strains may refer to a single microbial species in the plural form.

[0045] As used herein, "complexity" refers to the number of strains in a community, regardless of abundance. A community containing 50 strains is more complex than a community containing 15 strains. As used herein, "high complexity" refers to a community having at least 40 strains of a defined microbial strain. In some embodiments, a high complexity community is between 40-500, between 40-400, between 40-300, between 40-200, between 40-150, between 40-140, between 40-130, between 40-120, between 40-110, between 40-100, between 50-500, between 50-400, between 50-300, between 50-200, between 50-150, between 50-140, between 50-160, between 50-180, between 50-190, between 50-210, between 50-220, between 50-230, between 50-240, between 50-250, between 50-260, between 50-270, between 50-280, between 50-290, between 50-310, between 50-320, between 50-330, between 50-340, between 50-350, between 50-360, between 50-370, between 50-380, between 50-390, between 50-410, between 50-420, between 50-430, between 50-440, between 50-450, between 50-460, between 50-480, between 50-490, between 50-510, between 50-520, between 50-53 Between 30, Between 50-120, Between 50-110, Between 50-100, Between 60-500, Between 60-400, Between 60-300, Between 60-200, Between 60-150, Between 60-140, Between 60-130, Between 60-120, Between 60-110, Between 60-100, Between 70-500, Between 70-400, Between 70-300, Between 70-200, Between 70-150 , between 70-140, between 70-130, between 70-120, between 70-110, between 70-100, between 80-500, between 80-400, between 80-300, between 80-200, between 80-150, between 80-140, between 80-130, between 80-120, between 80-110, between 80-100, between 90-500, between 90-400, between 90-300, between 90- between 200, between 90 and 150, between 90 and 140, between 90 and 130, between 90 and 120, between 90 and 110, between 90 and 100, between 100 and 500, between 100 and 400, between 100 and 300, between 100 and 200, between 100 and 150, between 100 and 140, between 100 and 130, between 100 and 120, or between 100 and 110. 3.1. Cultivation of microbial strains and communities

[0046] As used herein, "culture" (and grammatical variations thereof, e.g., "cultured" and "culturing") refers to the maintenance and / or growth of a microbial strain or community in liquid medium or on solid medium. For example, in some embodiments, cultures of purchased microbial strains are performed according to the manufacturer's instructions.

[0047] As used herein, "aliquot" refers to an in vitro population of bacteria that is physically separated from other populations for storage, culture, analysis, etc. An "aliquot" can refer to a separate population in a container, compartment, tube, well of a multi-well plate, a clonal emulsion, a stock of isolates, or can be a mixture of strains, e.g., an artificial community or a defined gut microbial community.

[0048] In certain embodiments, the microbial strain or microbial community is maintained or grown in a specially formulated medium, such as the universal growth medium described in Table 1 below. Table 1 [Table 1-1] [Table 1-2] 3.2. Engraftment

[0049] As used herein, "engraftment" (and grammatical variations thereof, e.g., "engraft") refers to the ability of a microbial strain or microbial community to establish in one or more microenvironments of the intestine of an animal. Operationally, a microbial strain or microbial community is "engrafted" if evidence of this establishment can be obtained following administration. In some embodiments, this evidence can be obtained by molecular identification (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S In some embodiments, the sample is obtained by rRNA sequencing, rRNA sequencing, or genomic sequencing). In some embodiments, the sample is a fecal sample. In some embodiments, the sample is a biopsy taken from the intestine of the animal (e.g., from a site along the gastrointestinal tract of the animal). Engraftment can be transient or persistent. In some embodiments, transient engraftment means that the microbial strain or microbial community is no longer detectable in the administered animal after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, or about 2 years.

[0050] As used herein, "substantial engraftment" refers to being able to demonstrate evidence of engraftment of at least 70% of the administered defined microbial strains at a defined time point after administration of a microbial community (e.g., a high complexity defined gut microbial community) to an animal (e.g., in some embodiments, a gnotobiotic mouse) but prior to exposure to any microbial community (e.g., human fecal community microbial exposure). For example, in some embodiments, substantial engraftment is achieved when at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or 100% of the administered defined microbial strains are demonstrable. In some embodiments, such evidence is obtained by metagenomic analysis of fecal samples obtained from the mice. In some embodiments, "substantial engraftment" is achieved when the intended metabolic phenotype is demonstrably present in the recipient after administration and prior to microbial exposure. In some embodiments, the defined time point is between 1 week and 52 weeks.For example, in some embodiments, the defined time points are between 1 week and 48 weeks, between 1 week and 42 weeks, between 1 week and 36 weeks, between 1 week and 30 weeks, between 1 week and 24 weeks, between 1 week and 18 weeks, between 1 week and 12 weeks, between 1 week and 10 weeks, between 1 week and 8 weeks, between 1 week and 6 weeks, between 1 week and 4 weeks, between 1 week and 2 weeks, between 2 weeks and 52 weeks, between 2 weeks and 48 weeks, between 2 weeks and 36 weeks, between 2 weeks and 30 weeks, between 2 weeks and 24 weeks, between 2 weeks and 18 weeks, between 2 weeks and 12 weeks, between 2 weeks and 10 weeks, between 2 weeks and 8 weeks, between 2 weeks and 6 weeks, between 2 weeks and 4 weeks, between 4 weeks and 52 weeks, between 4 weeks and 48 weeks, between 4 weeks and 42 weeks. , between 4 weeks and 36 weeks, between 4 weeks and 30 weeks, between 4 weeks and 24 weeks, between 4 weeks and 18 weeks, between 4 weeks and 12 weeks, between 4 weeks and 10 weeks, between 4 weeks and 8 weeks, between 4 weeks and 6 weeks, between 6 weeks and 52 weeks, between 6 weeks and 48 weeks, between 6 weeks and 42 weeks, between 6 weeks and 36 weeks, between 6 weeks and 30 weeks, between 6 weeks and 24 weeks, between 6 weeks and 18 weeks, between 6 weeks and 12 weeks, between 6 weeks and 10 weeks, between 6 weeks and 8 weeks, between 8 weeks and 52 weeks, between 8 weeks and 48 weeks, between 8 weeks and 42 weeks, between 8 weeks and 36 weeks, between 8 weeks and 30 weeks, between 8 weeks and 24 weeks, between 8 weeks and 18 weeks, between 8 weeks and 12 weeks, or between 8 weeks and 10 weeks. 3.3. Stability

[0051] As used herein, "human fecal community microbial exposure" refers to the administration of a human fecal sample into the intestine of an animal that has been previously colonized with a microbial community, e.g., a defined gut microbial community of high complexity.

[0052] In some embodiments, the stability of a community refers to the ability of the defined microbial strains that compose the community to persist (i.e. remain engrafted) in the intestine of an animal after microbial exposure. In some embodiments, given sufficient time to allow colonization of the microbial exposure strains in the intestine of an animal engrafted with a high complexity defined gut microbial community, a stable community can be defined as a stable community when at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the defined microbial strains are detectable by metagenomic analysis. For example, in some embodiments, metagenomic analysis includes whole genome shotgun sequencing analysis.

[0053] In some embodiments, stability can be demonstrated for a time period ranging from at least 1 week to 52 weeks. For example, in some embodiments, stability can be demonstrated for a time period ranging from at least 1 week to 48 weeks, 1 week to 42 weeks, 1 week to 36 weeks, 1 week to 30 weeks, 1 week to 24 weeks, 1 week to 18 weeks, 1 week to 12 weeks, 1 week to 10 weeks, 1 week to 8 weeks, 1 week to 6 weeks, 1 week to 4 weeks, 1 week to 2 weeks, 2 weeks to 52 weeks, 2 weeks to 48 weeks, 2 weeks to 36 weeks, 2 weeks to 30 weeks, 2 weeks to 24 weeks, 2 weeks to 18 weeks, 2 weeks to 12 weeks, 2 weeks to 10 weeks, 2 weeks to 8 weeks, 2 weeks to 6 weeks, 2 weeks to 4 weeks, 4 weeks to 52 weeks, 4 weeks to 48 weeks, 4 weeks to 42 weeks, 4 weeks to 3 weeks, 4 weeks to 52 ... The period may be demonstrated in a range of times, such as between 6 weeks, between 4 weeks and 30 weeks, between 4 weeks and 24 weeks, between 4 weeks and 18 weeks, between 4 weeks and 12 weeks, between 4 weeks and 10 weeks, between 4 weeks and 8 weeks, between 4 weeks and 6 weeks, between 6 weeks and 52 weeks, between 6 weeks and 48 weeks, between 6 weeks and 42 weeks, between 6 weeks and 36 weeks, between 6 weeks and 30 weeks, between 6 weeks and 24 weeks, between 6 weeks and 18 weeks, between 6 weeks and 12 weeks, between 6 weeks and 10 weeks, between 6 weeks and 8 weeks, between 8 weeks and 52 weeks, between 8 weeks and 48 weeks, between 8 weeks and 42 weeks, between 8 weeks and 36 weeks, between 8 weeks and 30 weeks, between 8 weeks and 24 weeks, between 8 weeks and 18 weeks, between 8 weeks and 12 weeks, or between 8 weeks and 10 weeks.

[0054] In other embodiments, the stability of a community refers to the property that the defined microbial strains that make up the community maintain a metabolic phenotype over a period of time or after microbial exposure.For example, in some embodiments, the defined microbial strains that make up the community can maintain a metabolic phenotype for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 1 year, at least 1.5 years, or at least 2 years.

[0055] In some embodiments, a stable community can be defined as a stable community if the defined microbial strains that make up the community maintain one or more metabolic phenotypes of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, or propionate production over a period of time or after microbial exposure.

[0056] As used herein, "dropout" refers to the phenomenon that the microbial strains in the microbial community do not stably settle after administration into the intestine of an animal.For example, in some embodiments, the microbial community is stable when up to 10% of the defined microbial strains drop out after microbial exposure.In some embodiments, the microbial community is stable when up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2% or up to 1% of the defined microbial strains drop out after microbial exposure.

[0057] As used herein, "jump-in" refers to the phenomenon in which a microbial strain not present in the microbial community at the time of administration to an animal stably colonizes one or more microenvironments of the animal's intestine and becomes part of the colonized microbial community. In some embodiments, the microbial strain that jumps in is derived from the animal's gut commensal repertoire, fecal community microbial exposure, or administration into the animal's intestine after the first administration of the microbial community. For example, in some embodiments, the microbial community is stable when up to 10% of the new strains are due to microbial exposure (e.g., human fecal community microbial exposure). In some embodiments, the microbial community is stable when up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% of the new strains are due to microbial exposure. 4. Metagenomic Analysis and Molecular Identification

[0058] As used herein, "metagenomic analysis" refers to the use of massively parallel sequencing to analyze the microbiome or defined gut microbial community. As used herein, metagenomic analysis includes, but is not limited to, whole genome sequencing (e.g., in some embodiments, whole genome shotgun sequencing), ribosomal gene sequencing, rRNA sequencing or other sequencing-based methods. See, for example, Thomas et al., 2012, "Metagenomics - A guide from sampling to data analysis," Microbial Informatics and Experimentation 2(1):3; Qin et al., 2009. "A human gut microbial gene catalogue established by metagenomic sequencing," Nature 464 (7285): 59-65. For example, in some embodiments, metagenomic sequence read data (i.e., sequence fragments) obtained from the sequencing method are mapped against a comprehensive database of complete sequenced genomes of all defined microbial strains that constitute the gut community.

[0059] As used herein, "molecularly identified" (and grammatical variations thereof, e.g., "molecular identification") refers to the characterization of a microbial species for unique identification. In some embodiments, molecular identification can be 16S rRNA sequencing, whole genome sequencing, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), or similar analytical assays capable of distinguishing one microbial species from another. In some embodiments, species identification is made at the level of strain identification. In some embodiments, strain identification is achieved by whole genome shotgun metagenomic sequencing. As used herein, whole genome shotgun metagenomic sequencing refers to a method of sequencing polynucleotides in parallel from multiple genomic regions from a complex sample containing multiple microbial species with high sequence coverage. 5. In vitro and metabolic phenotypes

[0060] As used herein, "in vitro phenotype" refers to a characteristic of a microbial community that can be measured in vitro, e.g., metabolic phenotype. In one embodiment, the microbial community is recovered from the intestine of an animal. In one embodiment, the microbial community is recovered from a stool sample. In one embodiment, the microbial community is an artificial community or a high-complexity defined gut microbial community.

[0061] "Metabolic phenotype" is a characteristic of a microbial strain or microbial community. In one aspect, metabolic phenotype refers to the ability of a microbial strain or microbial community to transform one or more first compound(s) into one or more second compound(s). In one example, a first compound is enzymatically converted by a microorganism or community into a second compound, and the metabolic phenotype is an increase in the amount of the second compound. In some embodiments, the metabolic phenotype includes mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methane production, acetate production, hydrogen production, and propionate production. For example, in some embodiments, one or more of the defined microbial strains of the high complexity defined gut microbial community have at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all of the above metabolic phenotypes.

[0062] In some embodiments, the metabolic phenotype is selected from the group consisting of fructan, inulin, glucuronoxylan, arabinoxylan, glucomannan, beta mannan, dextran, starch, arabinan, xyloglucan, galacturonan, beta glucan, galactomannan, rhamnogalacturonan I, rhamnogalacturonan II, arabinogalactan, mucin O-linked glycan, yeast alpha mannan, yeast beta glucan, chitin, alginate, porphyrin, laminarin, carrageenan, agarose, alternan, levan, xanthan gum, galactooligosaccharides, hyaluronan, chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, phenylalanine, tyrosine, tryptophan, leucine, including the ability to convert valine, isoleucine, glycine, proline, asparagine, glutamine, aspartic acid, glutamic acid, cysteine, lysine, arginine, serine, methionine, alanine, arginine, histidine, ornithine, citrulline, carnitine, hydroxyproline, cholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholesterol, cinnamic acid, coumaric acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, chlorogenic acid, catechin, epicatechin, gallic acid, pyrogallol, catechol, quercetin, myricetin, campherol, luteolin, apigenin, naringenin, and / or hesperidin. For example, in some embodiments, the combined multiple defined microbial strains of the high complexity defined gut microbial community are capable of metabolizing at least 2, at least 4, at least 8, at least 12, at least 24, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or all of the above compounds. 6. Microbial Community Backfill

[0063] Described herein are "backfill" methods for generating high-complexity defined gut microbial communities. Backfill methods include "in vitro backfill" and "in vivo backfill". In vitro backfill and in vivo backfill may be used in combination as described below. In some embodiments, only in vitro backfill is used to generate the community. In some embodiments, only in vivo backfill is performed to generate the community. Also described herein are compositions for use in or generated by such backfill processes.

[0064] For convenience, the term "backfill" will be used to describe the process of backfilling in vitro or in vivo, and the term "backfill community" will refer to the community generated by the backfill process. 6.1 Generation of complex communities by in vitro backfilling

[0065] In one aspect, the invention involves generating a complex microbial community by in vitro backfilling. The community generated by one or more rounds of in vitro backfilling may be used as starting stock for one or more rounds of in vivo backfilling. 6.2 Microbial Pantry

[0066] As discussed below, the backfill process involves several steps of preparing an artificial community by combining several individually selected bacterial strains in the same aliquot. The inventors designed an initial collection of 109 organisms found in the human gut (including 104 bacterial strains that are highly prevalent in the population and 4 archaeal strains). In one aspect, the present invention provides a "microbial pantry", an array, e.g., a multi-well plate, of aliquots containing clonal isolates, as a useful tool for carrying out the backfill method, where a significant proportion of the strains in Table 2, e.g., at least 80, at least 90, at least 95, or at least 100, are contained in the array aliquot. In some embodiments, the array is a multi-well plate. Also, a system is contemplated in which any combination of individual strains in the "pantry" can be collected and combined in an aliquot by automatic robotic control. Thus, in one aspect, the present invention includes a system including an array and a robot under computer control for transferring bacteria. The term "microbial pantry" can also refer to a collection of clonal aliquots (e.g., tubes) that contain at least a substantial portion of the strains listed in Table 2 together, even if not physically associated in an array, but the aliquots are in the same location so that any combination of strains can be obtained. The microbial pantry is typically stored frozen until use. In some cases, the microorganisms are provided as spores. Table 2: Example strains from the microbial pantry [Table 2-1] [Table 2-2] [Table 2-3]

[0067] In addition to the strains listed in Table 2, it is contemplated that other bacterial strains (typically anaerobes or facultative anaerobes) may be used in the backfill method, including non-naturally occurring genetically modified organisms. Exemplary genetic modifications include, but are not limited to, mutations or knockouts of enzyme-encoding genes and expression of heterologous genes. 6.3 Primary Backfill Crowd

[0068] Backfilling is an iterative process. A "first backfill community" is prepared by combining strains of the "scaffold community" with the "backfill strain". Broadly speaking, and without intending to be bound by a particular mechanism, the scaffold community is a combination of strains selected to produce a desired metabolic phenotype. The backfill strain is a combination of strains selected to include strains that contribute to the in vitro stability of the first backfill community and contribute to the stability of the resulting transplantable community in the human intestine. Without intending to be bound by a particular mechanism, it is believed that the backfilling process increases the complexity of the community, and that communities with higher complexity tend to inhabit a more microenvironment in the intestine, making the community more stable. 6.4 Scaffolding Crowds

[0069] The scaffold community includes multiple strains common in the human gut microbiome. A given scaffold community typically includes 5-100 strains, usually 10-30 strains. The scaffold community may include one or more strains listed in Table 2, for example, at least 5 strains, at least 10 strains, at least 20 strains, at least 30 strains, etc., listed in Table 2. In some approaches, at least 50%, 75%, 90% or all of the strains in the scaffold community are selected from Table 2.

[0070] The scaffold community is selected to exhibit a desired phenotype, typically a desired metabolic phenotype. The "metabolic phenotype" of a community refers to the production or consumption of metabolites by the community, as described above. An exemplary metabolic phenotype is the ability to increase or decrease the concentration of one or more compounds in an environment as a result of a microbial metabolic process. For example, a scaffold community comprising Clostridium sporogenes may consume phenylalanine to produce tyrosine, in which case the metabolic phenotype may be "producing tyrosine". Similarly, a community comprising Proteus mirabilis in an environment containing urea may decrease the concentration of urea and increase the concentration of ammonia, and a community comprising Bacillus subtilis in an environment containing sucrose may decrease the concentration of sucrose and increase the concentration of glucose. However, importantly, such a simple illustration grossly oversimplifies the metabolic processes occurring in a microbial ecosystem. For example, the metabolic products of a first member of a microbial community may be metabolic substrates for a second member of the community, or the metabolic products of one member of a microbial community may be transcriptional activators in another microorganism, or may be toxic to other microorganisms.In complex microbial ecosystems that contain hundreds of different strains, even if the various entities present are known, it is impossible to use current methods to accurately predict the network of interactions between strains, metabolites, and environmental factors in a particular microbial ecosystem.In addition, unless or until a microbial ecosystem is in a homeostatic state, the combination of strains in a population may be unstable and change in an unpredictable way, which may change the metabolic phenotype of the community. 6.5 Addition of backfill strains to scaffold populations to create the first in vitro backfill population

[0071] To generate the first in vitro backfill community, the designed scaffold community is supplemented with additional microbial strains, referred to as "backfill strains." For example, each scaffold community may be combined with an additional strain of 35-495 species. In some embodiments, each scaffold community may be combined with between 40-400, between 40-300, between 40-200, between 40-150, between 40-140, between 40-130, between 40-120, between 40-110, between 40-100, between 50-400, between 50-300, between 50-200, between 50-150, between 50-140, between 50-130, between 50-160, between 50-170, between 50-180, between 50-190, between 50-210, between 50-220, between 50-240, between 50-260, between 50-280, between 50-290, between 50-310, between 50-320, between 50-330, between 50-340, between 50-350, between 50-410, between 50-420, between 50-430, between 50-440, between 50-450, between 50-550, between 50-560, between 50-600, between 50-650, between 50-700, between 50-800, between 50-900, between 50-110, between 50-140, between 50-150, between 50-160, between 50-180, between 50-190, between 50-2 Between 0-120, Between 50-110, Between 50-100, Between 60-400, Between 60-300, Between 60-200, Between 60-150, Between 60-140, Between 60-130, Between 60-120, Between 60-110, Between 60-100, Between 70-500, Between 70-400, Between 70-300, Between 70-200, Between 70-150, Between 70-500, Between 70-400, Between 70-300, Between 70-200, Between 70-150, Between 70-400, Between 70-500, Between 70-400, Between 70-300, Between 70-200, Between 70-150, Between 70-400, Between 70-500, Between 70-500, Between 70-600, Between 70-700, Between 70-800, Between 70-900, Between 70-100, Between 70-120, Between 70-140, Between 70-15 ... Between 0-140, Between 70-130, Between 70-120, Between 70-110, Between 70-100, Between 80-400, Between 80-300, Between 80-200, Between 80-150, Between 80-140, Between 80-130, Between 80-120, Between 80-110, Between 80-100, Between 90-400, Between 90-300, Between 90-200, Between 9 ...300, Between 90-400, Between 90-500, Between 90-600, Between 90-700, Between 90-800, Between 90-900, Between 90-100, Between 90-110, Between 90-100, Between 90-120, Between 90-130, Between 90-140, Between 90-130, Between 90-120, Between 90-110, Between 90-100, Between 90-140, Between 90-200, Between 90-300, Between 90-400, Between 90-500 Between 0-150, between 90-140, between 90-130, between 90-120, between 90-110, between 90-100, between 100-400, between 100-300, between 100-200, between 100-150, between 100-140, between 100-130, between 100-120, or between 100-110 defined microbial strains. The backfill strains and the scaffold community strains may be combined in any order. For example, the backfill strains may be added to all of the scaffold community strains in a single batch. Alternatively, a subset of the scaffold community strains may be combined with a subset of the backfill strains in any desired order. 6.6 Parallel Backfill Crowds

[0072] The in vitro backfill method is carried out by testing many different strains and combinations in parallel according to the methods disclosed herein, as described in more detail below. In principle, a single first in vitro backfill strain can be generated by combining a single scaffold community with a backfill strain, but the robustness of the method arises in part from the parallel processing of multiple communities. Typically, multiple first in vitro backfill communities designed to exhibit a given metabolic phenotype are generated by combining scaffold and backfill communities (e.g., typically 2-100 communities, generally at least 5, at least 10 or at least 15 communities). In one approach, multiple aliquots of one scaffold community are used. In one approach, multiple different scaffold communities are used, where the communities are designed for the same metabolic phenotype, but with different (sometimes only slightly different) combinations of strains. Each approach may use one combination of backfill strains, or multiple different combinations of backfill strains. Thus, in an in vitro backfill process, multiple primary backfill populations can be created, grown, and assayed in parallel.

[0073] The number of different first backfill populations assayed in parallel can range from 2 to 100 or more. Typically, the number is greater than 5, greater than 10, greater than 25, greater than 50, or greater than 100. 6.7 In vitro backfill community cultivation

[0074] The first backfill population, as well as subsequent in vitro backfill populations (below), are cultured for a period of time and then evaluated as described below. Strains may be cultured for 2 hours to 10 days, although longer or shorter times may be used. For example, backfill populations may be cultured for 1 to 72 hours, e.g., 12 to 72 hours, 12 to 48 hours, or 24 to 48 hours. Typically, strains are cultured in an environment that mimics the temperature (e.g., 36 to 38°C) and low pO2 (e.g., under anaerobic conditions) of the human intestine. Preferably, a single universal culture medium is used, which may be designed to approximate conditions encountered in the mammalian (e.g., human) intestine. 6.8 Evaluation and Ranking of In Vitro Backfill Communities

[0075] At the end of the culture period, or multiple times during the culture period, one or more traits of the first backfill population as well as subsequent in vitro backfill populations can be evaluated. By way of illustration and not limitation, exemplary traits that can be evaluated include metabolic phenotype and antibiotic resistance. 6.9 Evaluation of stock composition

[0076] The strain composition of the backfill community can be determined at the end of the culture period or at any time during the culture period as desired. The strain composition can be determined by metagenomic analysis, by quantitative evaluation, e.g., qPCR, by microbiological techniques, e.g., colony counting, or by a combination of methods. In one embodiment, the abundance or relative proportion of individual strains can be measured. 6.10 Assessing changes in strain composition

[0077] By determining the strain composition of the community at various time points, changes in composition can be detected. The inventors observed that some strains "drop out" during culture and / or backfilling in vivo. The changes in strain composition over different rounds or iterations of backfilling in vitro or in vivo (discussed below) can be used as a measure of "community composition stability", i.e., stability as defined above. 6.11 Metabolic Phenotype Assessment

[0078] The metabolic phenotype of the backfill community can be determined at the end of the culture period or during the culture period. The metabolic phenotype can be assayed in any suitable manner based on the desired phenotype. For example, in one approach, one or more first compounds are combined with the community and the conversion of the first compound(s) to the second compound(s) is measured over time or at the end time. Detection and measurement of compounds or other attributes can be performed by any of a variety of methods. For example, mass spectrometry, liquid chromatography, immunoassays (ELISA), radiolabeled metabolite tracking, etc. can be used to detect compounds produced or consumed by the community. The assay can be performed under conditions that mimic the conditions of the mammalian (e.g., human) gut or across a number of conditions that mimic the variation in the gut of individuals of the population.

[0079] Changes in metabolic phenotype across different rounds or iterations of in vitro or in vivo backfilling (discussed below) can be used as a measure of "community phenotype stability." 6.12 Other Evaluations

[0080] The backfill community may also be tested for antibiotic susceptibility or resistance, contamination, etc. In some cases, the backfill community may be exposed to pathogens or other microorganisms to determine, for example, whether the addition of a pathogen perturbs or overpopulates the community. In some cases, the backfill community may be introduced into the intestines of humanized mice to determine whether the community can replace the gut microbiome. 6.13 Crowd Ranking

[0081] The first and subsequent backfill communities may be ranked according to the traits evaluated, e.g., metabolic phenotype. For example, if the desired community phenotype is the production of metabolite X under defined conditions, the ability of the community to produce X, the rate at which X is produced, or other kinetic measures, etc., may be measured, and backfill communities with a more robust desired phenotype may be ranked higher than communities in which the desired phenotype is absent or less robust. Multiple traits or criteria may be considered and assigned equal or unequal weights and used in the ranking. 6.14 Backfill Population Selection

[0082] As described above, the backfill communities may be ranked according to any combination of attributes and compared in any manner. In one approach, the highest ranked backfill community or communities are selected for further processing. In one approach, the highest ranked communities are selected for further processing. In one approach, 1%, 5%, 10% or 25% of the highest ranked communities are processed for further development. In one approach, communities exhibiting attributes above a predetermined threshold may be selected for further processing. Those communities not selected may be discarded.

[0083] The backfill population selected for further processing may be referred to as the "selected backfill population." 6.15 Further Processing of Subsequent Backfill Crowds

[0084] The selected (highest ranked) first backfill community or communities may be further processed in subsequent iterations or rounds of the in vitro backfill process. In one approach, the selected first backfill communities are processed in a manner similar to the processing of the scaffold community. In some embodiments, each selected first backfill community is split into multiple aliquots for parallel processing, and a small number of backfill strains (e.g., 1-50 strains) are added to each aliquot, thereby generating a "subsequent backfill community." The backfill strains added to each aliquot may not be identical for all aliquots of the first backfill community, but rather different combinations and complexities of backfill strains may be added. The process of adding backfill strains to a backfill community (e.g., a first backfill community) to generate a subsequent backfill community may be referred to as "challenging" or "evolving" the community.

[0085] The subsequent backfill populations are cultured for a period of time (the "culture period"), and at the end of the culture period, or multiple times during the culture period, one or more attributes of the subsequent populations are evaluated as described above, and the subsequent populations are ranked for additional iterations or rounds of further treatment. The attributes evaluated in one round of treatment and used for ranking may be the same or different from the attributes evaluated in previous or subsequent rounds. 6.16 Iteration

[0086] When developing complex communities for transplantation, multiple iterations of the backfill process may be performed. As used in this context, the generation of a first backfill community is the first iteration, and subsequent iterations are used to generate subsequent backfill communities, which are represented by ordinal numbers (second backfill community, third backfill community, etc.). As used in this context, a second or subsequent "iteration" includes the process of (1) adding at least one backfill strain to an existing backfill population to generate a next generation population, (2) culturing the next generation population, and (3) optionally characterizing the population.

[0087] The number of iterations to generate subsequent backfill populations (i.e., not including the first backfill population) can range from 1 to 20. Typically, the number of iterations ranges from 5 to 10 iterations. Generally, at least 1, 2, 3, 4, 5, 6, or 7 iterations are used to generate subsequent in vitro backfill populations.

[0088] As described above, the selected backfill community is divided into multiple aliquots, each of which can be combined with one or more backfill strains (e.g., if not all aliquots receive the same backfill strain). It may also be useful to describe the lineage of the community. In any subsequent iterations of backfill, communities generated from the same selected backfill community are referred to as "sibling communities" of each other and as "descendants" of the selected backfill community. The selected backfill community can be referred to as the "ancestor" of the descendant community. 6.17 Generation of transplantable populations by in vivo backfilling

[0089] After the final iteration of in vitro backfilling, one or more of the subsequent backfill populations may be identified as having desirable traits (e.g., a desired metabolic phenotype) and may be used as the first in vivo backfill population. The in vivo backfilling process coincides in some respects with the in vitro process described above. Many of the in vivo backfilling steps are identical or similar to the corresponding in vitro steps discussed above. The main differences are: - the first in vivo backfill community is not a scaffold community, but is typically a community generated by in vitro backfill; - engrafting the backfill community into a non-human animal (typically a gnotobiotic mouse) without culturing it in vitro; and - Exposing or evolving the engrafted backfill community by combining it with human fecal transplant material containing a complex mixture of strains. Backfill strains can also be administered as needed.

[0090] Similar to the in vitro method, multiple primary in vivo backfill populations can be developed in parallel, as described in more detail below. Thus, for example and not by way of limitation, one approach to backfilling in vivo includes the following steps: i. engrafting the selected in vitro backfill community into the intestine(s) of one or more mice or other non-human animals; iia. introducing human fecal transplant material into the intestine(s) of one or more mice (i.e., exposing the engrafted population) before or after step (i); iib. Optionally, a backfill strain (e.g., from a microbiome pantry) may also be administered to one or more mice; iii. maintaining the mouse or mice for a period to allow engraftment and for the introduced strain to colonize the intestine to produce a "gut community"; iv. assessing one or more characteristics of the gut community, including composition (i.e., the presence of strains that "jump in" or "drop out" relative to the in vitro backfill community engrafted in step (i)); v. Optionally, ranking the gut communities and selecting one or more gut communities for further processing; vi. for each selected intestinal community, engrafting the community into multiple mice; vii. exposing the mice of (vi) by introducing human fecal transplant material (as in step ii above) and performing additional repetitions of steps (ii)-(vi) until the desired endpoint; Includes.

[0091] Certain aspects of the in vivo backfilling method are described in further detail below.

[0092] In vivo backfilling is typically performed in gnotobiotic mice, humanized mice, or other mammals (e.g., monkeys, horses, cows, pigs, dogs, cats, etc.). Gnotobiotic mice are known in the art and are commercially available. In some embodiments, in vivo backfilling may be performed in human subjects.

[0093] The selected in vitro population or subsequent in vivo population can be engrafted into mice using standard methods, for example, gavage.

[0094] The engrafted community can be exposed to human fecal material when developing treatments for human patients. Fecal preparations from other species can be used in model systems or in developing treatments for veterinary purposes (see Hu, J et al., 2018, "Standardized Preparation for Fecal Microbiota Transplantation in Pigs," Front. Microbiol. 9:1328).

[0095] Stool donors can be selected or screened for particular characteristics, such as donor health.

[0096] The stool material is processed for transplantation using methods known in the art. In some cases, stool material from more than one individual is pooled for engraftment.

[0097] Fecal material may be introduced into the intestines of mice by gavage. Engrafted mice are housed under germ-free conditions for one day to four weeks. This interval may be referred to as the "colonization period."

[0098] At the end of the colonization period, or multiple times during the colonization period, one or more characteristics of the first backfill population as well as subsequent in vitro backfill populations can be evaluated.

[0099] For purposes of evaluation, the community may be recovered from the intestines of animals (e.g., mice) in any manner that maintains the integrity of the microbiome, including (1) recovery of strains from feces, (2) recovery of intestinal contents, and (3) surgical recovery of the intestine (e.g., by sacrificing the mice).

[0100] Community characteristics that can be assayed and suitable methods include those described for in vitro backfilling, including changes in strain composition, metabolic phenotype, and / or strain and phenotype stability.

[0101] In addition to analyzing the backfill population, the mice can be analyzed for phenotype. Characteristics include the general health and vitality of the mice, as well as changes in blood or other tissues, such as changes in plasma levels of metabolites, particularly metabolites associated with a desired metabolic phenotype.

[0102] The in vivo backfill population may be ranked according to the traits (e.g., metabolic phenotype) evaluated. Multiple traits or criteria may be considered and assigned equal or unequal weights and used in the ranking.

[0103] The selected (highest ranked) in vivo backfill population or populations may be further processed in subsequent iterations or rounds of the in vivo backfill process, ranging from 2 to 10 iterations (usually 2 to 5, often 2 to 4 iterations). After the final iteration of in vivo backfill, one or more subsequent in vivo backfill populations may be identified as suitable for use as therapeutics and may be referred to as "therapeutic backfill populations." 6.18 In vivo backfilling

[0104] For in vivo backfilling, one approach is to administer to a non-human animal a defined gut community that is generated by a series of steps including: 1. Obtaining a first defined microbial community having an in vitro phenotype. Typically, the first defined microbial community is a product of an in vitro backfill. The in vitro phenotype may be a metabolic phenotype. 2. Engrafting a defined microbial community in the gut of an animal, typically a mouse, e.g., a germ-free mouse. This engraftment step can be performed in parallel in multiple (i.e., two or more) animals. 3. Exposing the animal to a human fecal community (e.g., feces from a human). In this context, "exposure" means introducing a human fecal community into the intestine of an animal previously engrafted with a defined microbial community to mix the two communities. Alternatively, the two communities can be combined before engraftment and the mixture can be engrafted into the animal. The exposed engrafted animal is maintained for a sufficient time to establish a community in the intestine that includes microorganisms from both the human fecal community and the defined microbial community, which can be called a "gut community". The gut community may contain fewer or more strains than the defined microbial community. The gut community may include strains that originate from the human fecal community (strains that "jumped in"). The gut community may not include strains that were present in the defined microbial community (strains that "dropped out"). If more than one animal is exposed, they may be exposed to the same human fecal preparation or different human fecal preparations. In one approach, not all of the animals are exposed to the same human fecal community. 4. Performing metagenomic analysis to detect strains in the gut community and determining whether differences exist between the gut community and the defined community. If differences exist (strains jump in or drop out), prepare a new defined microbial community ("subsequent defined microbial community") (e.g., using strains from the microbial pantry and / or other sources). The subsequent defined microbial community is engrafted onto an animal (e.g., a previously unengrafted animal) and processed as discussed above as the first defined microbial community. Such steps can be repeated for multiple iterations. For example, they can be repeated 1, 2, 3, 4, 5 or 6 times (e.g., typically 1-4 times). 5. Performing one or more assays to confirm that the gut community retains the desired phenotype (i.e., a phenotype that provides a therapeutic benefit to the patient). Gut communities that do not retain the phenotype are discarded. In some approaches, multiple different gut communities can be ranked based on the results of the assay, for example, where communities that strongly express the phenotype are ranked higher. In some approaches, the higher ranked communities are processed further and the lower ranked communities are discarded. 6. If the defined microbial community is stable, e.g., the difference between the engrafted and exposed, jumped-in or dropped-out strains is minimal and retains the desired phenotype, the defined microbial community may be used as a therapeutic. In some approaches, the defined microbial community is considered stable if there is less than a threshold of jumped-in strains and / or less than a threshold of dropped-out strains. In some embodiments, the jump-in and drop-out thresholds are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 strains. In some embodiments, the jump-in and drop-out thresholds are independently selected from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the strains in the engrafted defined microbial community. 6.19 Variations

[0105] In certain embodiments, the first in vitro population (generated by combining the scaffold population with the backfill strain) can be engrafted into a mammal without an in vitro backfill process. 7. Generation of High-complexity Defined Gut Microbial Communities

[0106] In some embodiments, a high complexity defined gut microbial community can be produced by the steps of: i) combining a plurality of defined microbial strains; ii) engrafting the combined plurality of defined microbial strains in the gut of an animal to generate an engrafted animal; iii) exposing the engrafted animal to a human stool sample; iv) maintaining the exposed engrafted animal for a time sufficient for intestinal colonization of the animal with the microbial strains of the human stool sample, thereby generating a gut community in the gut of the animal; v) identifying the microbial strains of the gut community by metagenomic analysis; vi) identifying whether there are differences between the microbial strains that make up the gut community and the microbial strains that make up the combined plurality of defined microbial strains; and vii) determining whether there are differences between the microbial strains that make up the gut community and the combined plurality of defined microbial strains. If there is a significant difference between the microbial strains constituting the number of defined microbial strains, add one or more additional defined microbial strains that were not present in step i) to the combined multiple defined microbial strains or remove the defined microbial strains that were present in the combined multiple defined microbial strains of step i) to generate a modified combined multiple defined microbial strains, and repeat steps ii) to vi) in the non-engrafted animal using the modified combined multiple defined microbial strains as the combined multiple defined microbial strains, and if there is a minimal difference, the modified defined microbial community of the final step vii) can be generated by an in vivo backfill process that is a high complexity defined gut microbial community. In some embodiments, the defined microbial strains are selected for combination to form a multiple microbial strain for engraftment based on the metabolic phenotype of the microbial strains. Selection of defined microbial strains with known metabolic phenotypes can form a high complexity defined metabolic community with improved engraftment and / or stability in one or more gut microenvironments.

[0107] In some embodiments, the metabolic phenotypes may include, but are not limited to, mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. For example, in some embodiments, a high complexity defined metabolic community may have at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or all of the above metabolic phenotypes.

[0108] In certain embodiments, the metabolic phenotype is selected from the group consisting of fructan, inulin, glucuronoxylan, arabinoxylan, glucomannan, beta-mannan, dextran, starch, arabinan, xyloglucan, galacturonan, beta-glucan, galactomannan, rhamnogalacturonan I, rhamnogalacturonan II, arabinogalactan, mucin O-linked glycan, yeast alpha mannan, yeast beta-glucan, chitin, alginate, porphyrin, laminarin, carrageenan, agarose, alternan, levan, xanthan gum, galactooligosaccharides, hyaluronan, chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, phenylalanine, tyrosine, tryptophan, leucine, valine, isoleucine, The ability to convert glycine, proline, asparagine, glutamine, aspartic acid, glutamic acid, cysteine, lysine, arginine, serine, methionine, alanine, arginine, histidine, ornithine, citrulline, carnitine, hydroxyproline, cholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholesterol, cinnamic acid, coumaric acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, chlorogenic acid, catechin, epicatechin, gallic acid, pyrogallol, catechol, quercetin, myricetin, campherol, luteolin, apigenin, naringenin, or hesperidin into one or more other compounds may include, but is not limited to, the ability to convert glycine, proline, asparagine, glutamine, aspartic acid, glutamic acid, cysteine, lysine, arginine, serine, methionine, alanine, arginine, histidine, ornithine, citrulline, carnitine, hydroxyproline, cholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholesterol, cinnamic acid, coumaric acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, chlorogenic acid, catechin, epicatechin, gallic acid, pyrogallol, catechol, quercetin, myricetin, campherol, luteolin, apigenin, naringenin, or hesperidin into one or more other compounds. For example, in some embodiments, the combined multiple defined microbial strains may be capable of metabolizing at least 2, at least 4, at least 8, at least 12, at least 24, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or all of the above compounds.

[0109] The active ingredient in the strain of the fermentation strain is:Acidaminococcus fermentans DSM 20731、Acidaminococcus sp.D21、Akkermansia muciniphila ATCC BAA-835、Alistipes putredinis DSM 17216、Anaerofustis stercorihominis DSM 17244、Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.2_1_22 sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium adolescentis L2-32、Bifidobacterium breve. DSM 20213、Bifidobacterium catenulatum DSM 16992、Bifidobacterium longum infantis ATCC 55813、Bifidobacterium pseudocatenulatum DSM 20438、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Bryantella formatexigens DSM 14469、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795, Clostridium bolteae ATCC BAA-613, Clostridium hathewayi DSM 13479, Clostridium hylemonae DSM 15053, Clostridium leptum DSM 753, Clostridium methylpentosum DSM 5476, Clostridium nexile DSM 1787, Clostridium saccharolyticum WM1 DSMZ 2544, Clostridium scindens ATCC 35704, Clostridium sp.L2-50, Clostridium sp.M62 / 1, Clostridium spiroforme DSM 1552, Clostridium sporogenes ATCC 15579, Collinsella aerofaciens ATCC 25986, Collinsella stercoris DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dialister invisus DSM 15470、Dorea formicigenerans ATCC 27755、Dorea longicatena DSM 13814、Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Lactobacillus ruminis ATCC 25644, Lactococcus lactis subsp.lactis Il1403 -> sub DSMZ 20729, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Olsenella uli DSM 7084、Parabacteroides distasonis ATCC 8503、Parabacteroides johnsonii DSM 18315、Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp.D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii L2-32, Ruminococcus flavefaciens FD 1, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum variabile DSM 15176, Veillonella dispar ATCC 17748, Veillonella sp.3_1_44 HM 64, and Veillonella sp.6_1_27 HM 49.

[0110] In certain embodiments, the high complexity defined gut microbial community is comprised of the following microbial strains: Acidaminococcus fermentans DSM 20731, Acidaminococcus sp. D21, Adlercreutzia equolifaciens DSM 19450, Akkermansia muciniphila ATCC 61163, and / or Acidaminococcus sp. D21. BAA-835、Alistipes finegoldii DSM 17242、Alistipes ihumii AP11、Alistipes indistinctus YIT 12060 / DSM 22520、Alistipes onderdonkii DSM 19147、Alistipes putredinis DSM 17216、Alistipes senegalensis JC50 / DSM 25460、Alistipes shahii WAL 8301 / DSM 19121、Anaerofustis stercorihominis DSM 17244、Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides rodentium DSM 26882、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis、ATCC. 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium breve、Bifidobacterium catenulatum DSM 16992、Bifidobacterium pseudocatenulatum DSM 20438、Bilophila wadsworthia ATCC 49260、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Blautia sp.KLE 1732(HM 1032)、Blautia wexlerae DSM 19850、Bryantella formatexigens DSM 14469、Burkholderiales bacterium 1_1_47、Butyricimonas virosa DSM 23226、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridiales bacterium VE202-03、Clostridiales bacterium VE202-14、Clostridiales bacterium VE202-27、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795, Clostridium bolteae ATCC BAA-613, Clostridium hathewayi DSM 13479, Clostridium hylemonae DSM 15053, Clostridium leptum DSM 753, Clostridium methylpentosum DSM 5476, Clostridium nexile DSM 1787, Clostridium saccharolyticum WM1 DSMZ 2544, Clostridium scindens ATCC 35704, Clostridium sp.ATCC 29733 VPI C48-50, Clostridium sp.L2-50, Clostridium sp.M62 / 1, Clostridium spiroforme DSM 1552, Collinsella aerofaciens ATCC 25986, Collinsella stercoris DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Intestinimonas butyriciproducens DSM 26588, Lactobacillus ruminis ATCC 25644、Megasphaera DSMZ 102144、Mitsuokella multacida DSM 20544、Odoribacter splanchnicus DSM 20712、Olsenella uli DSM 7084、Oscillibacter sp.KLE 1728、Parabacteroides distasonis ATCC 8503、Parabacteroides johnsonii DSM 18315、Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp.D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii ATCC, Ruminococcus flavefaciens FD 1, Ruminococcus gauvreauii DSM 19829, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum sp. 4_3_54A2FAA, Subdoligranulum variabile DSM 15176, and Veillonella dispar ATCC 17748, or a microbial strain selected from these.

[0111] In some embodiments, the method of generating a high complexity defined gut microbial community comprises culturing each of a plurality of defined microbial strains individually and then combining the defined microbial strains. In other embodiments, the method of generating a high complexity defined gut microbial community comprises culturing all of a plurality of defined microbial strains together. In yet other embodiments, the method of generating a high complexity defined gut microbial community comprises culturing one or more defined microbial strains individually to culture two or more of the defined microbial strains, and then combining the individually cultured and co-cultured defined microbial strains together. 8. Microbial communities for the treatment of dysbiosis or pathologies

[0112] The backfill community identified using the methods described herein can be used to treat patients by administration of a high complexity defined gut microbial community. Exemplary patients are those with dysbiosis or pathology. 8.1 Clostridium difficile infection

[0113] 8.1.1 Mouse model In some embodiments, when tested in a mouse model of C. difficile infection, the high complexity defined microbial communities of the invention reduce the number of C. difficile colony forming units (CFU) per μl of feces by at least 1-2 log, at least 2-3 log, at least 3-4 log, at least 4-5 log, or at least 5-6 log. In some embodiments, when tested in a mouse model of C. difficile infection, the high complexity defined microbial communities of the invention reduce the number of C. difficile colony forming units (CFU) per gram of feces by at least 1-2 log, at least 2-3 log, at least 3-4 log, at least 4-5 log, or at least 5-6 log. 8.1.2 Treatment of persistent C. difficile infection

[0114] In some embodiments, the high complexity defined gut microbial communities of the present invention can be used to treat animals with persistent C. difficile infections. For example, in some embodiments, the animal can be a mammal, more particularly a human.

[0115] In some embodiments, the method for generating a high complexity defined gut microbial community of the present invention for the treatment of persistent C. difficile infection includes the steps of: i) performing a C. difficile plate count on a fecal sample obtained from an animal with persistent C. difficile infection; ii) engrafting the high complexity defined gut microbial community in the intestine of the animal with persistent C. difficile infection to generate an engrafted infected animal; iii) maintaining the engrafted infected animal for a time sufficient for intestinal colonization with microbial strains of the high complexity defined gut microbial community, thereby generating an engrafted infected community in the intestine of the engrafted infected animal; iv) performing an additional C. difficile plate count on a fecal sample obtained from the engrafted infected animal; and v) determining whether the number of C. difficile CFUs obtained from the plate count of step iv) is greater than or equal to the number of C. difficile CFUs obtained from step i. and if the CFU count of C. difficile obtained from the plate count of step iv) is not significantly lower than the CFU count of C. difficile obtained from the plate count of step i), adding one or more additional defined microbial strains that were not present in step ii) to the high complexity defined gut microbial community to generate a modified high complexity defined gut microbial community and repeating steps i)-iv) in the animal with the non-engrafted, persistent C. difficile infection using the modified high complexity defined gut microbial community as the high complexity defined gut microbial community, wherein if there is a statistically significant reduction in the CFU count of C. difficile obtained from the plate count of step iv) compared to the CFU count of C. difficile obtained from the plate count of step i), the modified, stable defined gut microbial community of final step iv) may be the final high complexity defined gut microbial community.

[0116] In some embodiments, administration of an effective amount of a high complexity defined end-gut microbial community to an animal with persistent C. difficile infection effectively reduces the number of CFU of C. difficile per μl of feces in the treated animal. In some embodiments, administration of an effective amount of a high complexity defined end-gut microbial community to an animal with persistent C. difficile infection effectively reduces the number of CFU of C. difficile per gram of feces in the treated animal. 8.2 Bile acid metabolism and cholestatic disorders

[0117] In some embodiments, the high complexity defined gut microbial community significantly alters the profile and / or concentrations of bile acids present in an animal (e.g., mouse) fecal sample compared to an isogenic gnotobiotic control animal (e.g., an isogenic gnotobiotic control mouse).

[0118] For example, in some embodiments, the high complexity defined gut microbial communities of the present invention significantly alter the profiles and / or concentrations of Tβ-MCA, Tα-MCA, TUDCA, THDCA, TCA, 7β-CA, 7-oxo-CA, TCDCA, Tω-MCA, TDCA, α-MCA, β-MCA, ω-MCA, Muro-CA, d4-CA, CA, TLCA, UDCA, HDCA, CDCA, DCA and LCA in an animal (e.g., a mouse).

[0119] In some embodiments, the high complexity defined gut microbial communities of the present invention can be used to treat animals with cholestatic diseases, such as primary sclerosing cholangitis, primary biliary cholangitis, progressive familial intrahepatic cholestasis, or nonalcoholic steatohepatitis. For example, in some embodiments, the animal can be a mammal, more particularly a human. 9. Metabolite Modification

[0120] In some embodiments, the high complexity defined gut microbial community significantly alters the concentrations of metabolites present in an animal (e.g., mouse) urine sample compared to an isogenic gnotobiotic control animal (e.g., an isogenic gnotobiotic control mouse).

[0121] For example, in some embodiments, the high complexity defined gut microbial communities of the invention significantly alter concentrations of 4-hydroxybenzoic acid, L-tyrosine, 4-hydroxyphenylacetic acid, DL-p-hydroxyphenyllactic acid, p-coumaric acid, 3-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)pyruvic acid, indole-3-carboxylic acid, tyramine, L-phenylalanine, phenylacetic acid, 3-indoleacetic acid, DL-3-phenyllactic acid, L-tryptophan, DL-indole-3-lactic acid, phenylpyruvic acid, trans-3-indoleacrylic acid, 3-indolepyruvic acid, 3-indolepropionic acid, 3-phenylpropionic acid, trans-cinnamic acid, tryptamine, phenol, indole-3-carboxaldehyde, p-cresol, indole, 4-vinylphenol, or 4-ethylphenol. 10. Pharmaceutical Compositions

[0122] The product of the in vivo backfill process is a defined microbial community (e.g., a stable defined microbial community) with a known phenotype (e.g., a metabolic phenotype) that, when engrafted into a subject, confers an advantage to the subject.

[0123] Therapeutic backfill assemblages may be expanded and combined with excipients for oral administration (e.g., as a capsule), administration by naso / oral gavage, topical (e.g., by enema) or rectal (e.g., by colonoscopy) administration. Exemplary excipients include normal saline and others known in the art.

[0124] The present disclosure also provides pharmaceutical compositions containing an effective amount of microbial communities, for example, high complexity defined gut microbial communities. The compositions can be formulated for use in various delivery systems. Also, one or more physiologically acceptable excipient(s) or carrier(s) can be included in the composition for appropriate formulation. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0125] In some embodiments, the pharmaceutical compositions disclosed herein are directed to microbial communities of the invention, such as high complexity defined gut microbial communities, and to those that are not limited to carbohydrates (e.g., glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, fructose, maltose, cellobiose, lactose, deoxyribose, hexose); lipids (e.g., lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid ( 17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA) and tetracosane. acids (24:0); minerals (e.g., chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium); vitamins (e.g., vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin); buffering agents (e.g., sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate). preservatives (e.g., alpha-tocopherol, ascorbic acid, parabens, chlorobutanol, and phenol); binders (e.g., starch, pregelatinized starch, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, sugars, oligosaccharides);Lubricants (e.g., magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, Sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil); dispersing agents (e.g., starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose); disintegrants (e.g., corn starch (com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays such as bentonite, microcrystalline cellulose, alginic acid, sodium starch glycolate, gums such as agar, guar, locust bean gum, karaya, pectin, tragacanth, sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid); flavoring agents; sweeteners; and coloring agents.

[0126] In certain embodiments, the microbial communities of the invention, e.g., high complexity defined gut microbial communities, are administered orally as a lyophilized powder, capsule, tablet, troche, lozenge, granule, gel, or liquid. In some embodiments, the microbial communities of the invention, e.g., high complexity defined gut microbial communities, are administered as tablets or pills, which can be compressed, multi-compressed, multiply layered, and / or coated. 11. Dosage

[0127] In some embodiments, the microbial community of the present invention, e.g., a defined gut microbial community of high complexity, has a total abundance of 1×10 6 ~1X10 13 CFU, 1X10 6 ~1X10 12 CFU, 1X10 6 ~1X10 11 CFU, 1X10 6 ~1X10 10 CFU, 1X10 6~1X10 9 CFU、1X10 6 ~1X10 8 CFU、1X10 6 ~1X10 7 CFU、5X10 6 ~1X10 13 CFU、5X10 6 ~1X10 12 CFU、5X10 6 ~1X10 11 CFU、5X10 6 ~1X10 10 CFU、5X10 6 ~1X10 9 CFU、5X10 6 ~1X10 8 CFU、5X10 6 ~1X10 7 CFU、1X10 7 ~1X10 13 CFU、1X10 7 ~1X10 12 CFU、1X10 7 ~1X10 11 CFU、1X10 7 ~1X10 10 CFU、1X10 7 ~1X10 9 CFU、1X10 7 ~1X10 8 CFU、5X10 7 ~1X10 13 CFU、5X10 7 ~1X10 12 CFU、5X10 7 ~1X10 11 CFU、5X10 7 ~1X10 10 CFU、5X10 7 ~1X10 9 CFU、5X10 7 ~1X10 8 CFU、1X10 8 ~1X10 13 CFU、1X10 8 ~1X10 12 CFU、1X10 8 ~1X10 11 CFU、1X10 8 ~1X10 10 CFU、1X108 ~1X10 9 CFU, 5X10 8 ~1X10 13 CFU, 5X10 8 ~1X10 12 CFU, 5X10 8 ~1X10 11 CFU, 5X10 8 ~1X10 10 CFU, 5X10 8 ~1X10 9 CFU, 1X10 9 ~1X10 13 CFU, 1X10 9 ~1X10 12 CFU, 1X10 9 ~1X10 11 CFU, 1X10 9 ~1X10 10 CFU, 5X10 9 ~1X10 13 CFU, 5X10 9 ~1X10 12 CFU, 5X10 9 ~1X10 11 CFU, 5X10 9 ~1X10 10 CFU, 1X10 10 ~1X10 13 CFU, 1X10 10 ~1X10 12 CFU, 1X10 10 ~1X10 11 CFU, 5X10 10 ~1X10 13 CFU, 5X10 10 ~1X10 12 CFU, or 5X10 10 ~1X10 11 The microbial community is administered in a dosage form having a high complexity defined gut microbial community, for example a high complexity microbial community.

[0128] In some embodiments, the microbial communities of the invention, for example defined gut microbial communities of high complexity, may be present in a total amount of 0.1 ng to 500 mg, 0.5 ng to 500 mg, 1 ng to 500 mg, 5 ng to 500 mg, 10 ng to 500 mg, 50 ng to 500 mg, 100 ng to 500 mg, 500 ng to 500 mg, 1 μg to 500 mg, 5 μg to 500 mg, 10 μg to 500 mg, 50 μg to 500 mg, 100 μg to 500 mg, 500 μg to 500 mg, 1 mg to 500 mg, 5 mg to 500 mg, 10 mg to 500 mg, 50 mg~500mg, 100mg~500mg, 0.1ng~100mg, 0.5ng~100mg, 1ng~100mg, 5ng~100mg, 10ng~100mg, 50ng~100mg, 100ng~100mg, 500ng~500mg, 1μg~10 0mg, 5μg~100mg, 10μg~100mg, 50μg~100mg, 100μg~100mg, 500μg~100mg, 1mg~500mg, 5mg~100mg, 10mg~100mg, 50mg~100mg, 0.1ng~50mg, 0.5ng ~50mg, 1ng~50mg, 5ng~50mg, 10ng~50mg, 50ng~50mg, 100ng~50mg, 500ng~500mg, 1μg~50mg, 5μg~50mg, 10μg~50mg, 50μg~50mg, 100μg~50mg, 5 00μg~50mg, 1mg~500mg, 5mg~50mg, 10mg~50mg, 0.1ng~10mg, 0.5ng~10mg, 1ng~10mg, 5ng~10mg, 10ng~10mg, 50ng~10mg, 100ng~10mg, 500ng~50 0mg, 1μg~10mg, 5μg~10mg, 10μg~10mg, 50μg~10mg, 100μg~10mg, 500μg~10mg, 1mg~500mg, 5mg~10mg, 0.1ng~5mg, 0.5ng~5mg, 1ng~5mg, 5ng~5m g, 10ng~5mg, 50ng~5mg, 100ng~5mg, 500ng~500mg, 1μg~5mg, 5μg~5mg, 10μg~5mg, 50μg~5mg, 100μg~5mg, 500μg~5mg, 1mg~500mg, 0.1ng~1mg, 0.5ng~1mg、1ng~1mg、5ng~1mg、10ng~1mg、50ng~1mg、100ng~1mg、500ng~500mg、1μg~1mg、5μg~1mg、10μg~1mg、50μg~1mg、100μg~1mg、500μg~1mg、0.1ng~500μg、0.5ng~500μg、1ng~500μg、5ng~500μg、10ng~500μg、50ng~500μg、100ng~500μg、500ng~500μg、1μg~500μg、5μg~500μg、10μg~500μg、50μg~500μg、100μg~500μg、0.1ng~100μg、0.5ng~100μg、1ng~100μg、5ng~100μg、10ng~100μg、50ng~100μg、100ng~100μg、500ng~100μg、1μg~100μg、5μg~100μg、10μg~100μg、50μg~100μg、0.1ng~50μg、0.5ng~50μg、1ng~50μg、5ng~50μg、10ng~50μg、50ng~50μg、100ng~50μg、500ng~50μg、1μg~50μg、5μg~50μg、10μg~50μg、0.1ng~10μg、0.5ng~10μg、1ng~10μg、5ng~10μg、10ng~10μg、50ng~10μg、100ng~10μg、500ng~10μg、1μg~10μg、5μg~10μg、0.1ng~5μg、0.5ng~5μg、1ng~5μg、5ng~5μg、10ng~5μg、50ng~5μg、100ng~5μg、500ng~5μg、1μg~5μg、0.1ng~1μg、0.5ng~1μg、1ng~1μg、5ng~1μg、10ng~1μg、50ng~1μg、100ng~1μg、500ng~1μg、0.1ng~500ng、0.5ng~500ng、1ng~500ng、5ng~500ng、10ng~500ng、50ng~500ng、100ng~500ng、0.1ng~100ng、0.5ng~100ng、1ng~100ng、5ng~100ng、10ng~100ng、50ng~100ng、0.1ng~50ng、0.5ng~50ng、1ng~50ng、5ng~50ng、10ng~50ng、0.1ng~10ng、0.5ng~10ng、1ng~10ng、5ng~10ng、0.It is administered in a dosage form having a defined gut microbial community of 1 ng to 5 ng, 0.5 ng to 5 ng, 1 ng to 5 ng, 0.1 ng to 1 ng, 0.1 ng to 1 ng, or 0.1 ng to 0.5 ng of microbial community, e.g., high complexity.

[0129] In other embodiments, the microbial community of the present invention, e.g., a high complexity defined gut microbial community, is 1×10 6 ~1X10 13 CFU, 1X10 per day 6 ~1X10 12 CFU, 1X10 per day 6 ~1X10 11 CFU, 1X10 per day 6 ~1X10 10 CFU, 1X10 per day 6 ~1X10 9 CFU, 1X10 per day 6 ~1X10 8 CFU, 1X10 per day 6 ~1X10 7 CFU, 5X10 per day 6 ~1X10 13 CFU, 5X10 per day 6 ~1X10 12 CFU, 5X10 per day 6 ~1X10 11 CFU, 5X10 per day 6 ~1X10 10 CFU, 5X10 per day 6 ~1X10 9 CFU, 5X10 per day 6 ~1X10 8 CFU, 5X10 per day 6 ~1X10 7 CFU, 1X10 per day 7 ~1X10 13 CFU, 1X10 per day 7 ~1X10 12 CFU, 1X10 per day 7 ~1X10 11 CFU, 1X10 per day 7 ~1X10 10 CFU, 1X10 per day 7 ~1X109 CFU, 1X10 per day 7 ~1X10 8 CFU, 5X10 per day 7 ~1X10 13 CFU, 5X10 per day 7 ~1X10 12 CFU, 5X10 per day 7 ~1X10 11 CFU, 5X10 per day 7 ~1X10 10 CFU, 5X10 per day 7 ~1X10 9 CFU, 5X10 per day 7 ~1X10 8 CFU, 1X10 per day 8 ~1X10 13 CFU, 1X10 per day 8 ~1X10 12 CFU, 1X10 per day 8 ~1X10 11 CFU, 1X10 per day 8 ~1X10 10 CFU, 1X10 per day 8 ~1X10 9 CFU, 5X10 per day 8 ~1X10 13 CFU, 5X10 per day 8 ~1X10 12 CFU, 5X10 per day 8 ~1X10 11 CFU, 5X10 per day 8 ~1X10 10 CFU, 5X10 per day 8 ~1X10 9 CFU, 1X10 per day 9 ~1X10 13 CFU, 1X10 per day 9 ~1X10 12 CFU, 1X10 per day 9 ~1X10 11 CFU, 1X10 per day 9 ~1X10 10 CFU, 5X10 per day 9 ~1X10 13 CFU, 5X10 per day9 ~1X10 12 CFU, 5X10 per day 9 ~1X10 11 CFU, 5X10 per day 9 ~1X10 10 CFU, 1X10 per day 10 ~1X10 13 CFU, 1X10 per day 10 ~1X10 12 CFU, 1X10 per day 10 ~1X10 11 CFU, 5X10 per day 10 ~1X10 13 CFU, 5X10 per day 10 ~1X10 12 CFU, or 5X10 per day 10 ~1X10 11 Consumed in CFU ratios.

[0130] In other embodiments, the microbial communities of the invention, e.g., high complexity defined gut microbial communities, may be present in a range of concentrations between 0.1 ng and 500 mg per day, 0.5 ng and 500 mg per day, 1 ng and 500 mg per day, 5 ng and 500 mg per day, 10 ng and 500 mg per day, 50 ng and 500 mg per day, 100 ng and 500 mg per day, 500 ng and 500 mg per day, 1 μg and 500 mg per day, 5 μg and 500 mg per day, 10 μg and 500 mg per day, 5 ... 00mg, 100μg-500mg per day, 500μg-500mg per day, 1mg-500mg per day, 5mg-500mg per day, 10mg-500mg per day, 50mg-500mg per day, 100mg-500mg per day, 0.1ng-100mg per day, 0.5ng-100mg per day, 1ng-100mg per day, 5ng-100mg per day, 10ng-100mg per day, 50ng-100mg per day, 100ng-100m per day g, 500ng-500mg per day, 1μg-100mg per day, 5μg-100mg per day, 10μg-100mg per day, 50μg-100mg per day, 100μg-100mg per day, 500μg-100mg per day, 1mg-500mg per day, 5mg-100mg per day, 10mg-100mg per day, 50mg-100mg per day, 0.1ng-50mg per day, 0.5ng-50mg per day, 1ng-50mg per day, 5ng-50mg, 10ng-50mg per day, 50ng-50mg per day, 100ng-50mg per day, 500ng-500mg per day, 1μg-50mg per day, 5μg-50mg per day, 10μg-50mg per day, 50μg-50mg per day, 100μg-50mg per day, 500μg-50mg per day, 1mg-500mg per day, 5mg-50mg per day, 10mg-50mg per day, 0.1ng-10mg per day, 0.5ng-10mg, 1ng-10mg per day, 5ng-10mg per day, 10ng-10mg per day, 50ng-10mg per day, 100ng-10mg per day, 500ng-500mg per day, 1μg-10mg per day, 5μg-10mg per day, 10μg-10mg per day, 50μg-10mg per day, 100μg-10mg per day, 500μg-10mg per day, 1mg-500mg per day, 5mg-10 mg, 0.1ng-5mg per day, 0.5ng-5mg per day, 1ng-5mg per day, 5ng-5mg per day, 10ng-5mg per day, 50ng-5mg per day, 100ng-5mg per day, 500ng-500mg per day, 1μg-5mg per day, 5μg-5mg per day, 10μg-5mg per day, 50μg-5mg per day, 100μg-5mg per day, 500μg-5mg per day, 1mg-500mg per day , 0.1ng-1mg per day, 0.5ng-1mg per day, 1ng-1mg per day, 5ng-1mg per day, 10ng-1mg per day, 50ng-1mg per day, 100ng-1mg per day, 500ng-500mg per day, 1μg-1mg per day, 5μg-1mg per day, 10μg-1mg per day, 50μg-1mg per day, 100μg-1mg per day, 500μg-1mg per day, 0.1ng-500μg per day , 0.5ng-500μg per day, 1ng-500μg per day, 5ng-500μg per day, 10ng-500μg per day, 50ng-500μg per day, 100ng-500μg per day, 500ng-500μg per day, 1μg-500μg per day, 5μg-500μg per day, 10μg-500μg per day, 50μg-500μg per day, 100μg-500μg per day, 0.1ng-100μg per day, 0.5ng-100μg, 1ng-100μg per day, 5ng-100μg per day, 10ng-100μg per day, 50ng-100μg per day, 100ng-100μg per day, 500ng-100μg per day, 1μg-100μg per day, 5μg-100μg per day, 10μg-100μg per day, 50μg-100μg per day, 0.1ng-50μg per day, 0.5ng-50μg per day, 1ng-50μg per day, 5ng-50μg per day, 10ng-5 0μg, 50ng-50μg per day, 100ng-50μg per day, 500ng-50μg per day, 1μg-50μg per day, 5μg-50μg per day, 10μg-50μg per day, 0.1ng-10μg per day, 0.5ng-10μg per day, 1ng-10μg per day, 5ng-10μg per day, 10ng-10μg per day, 50ng-10μg per day, 100ng-10μg per day, 500ng-10μg per day, 1μg-10μg per day, 5μg- 10μg, 0.1ng-5μg per day, 0.5ng-5μg per day, 1ng-5μg per day, 5ng-5μg per day, 10ng-5μg per day, 50ng-5μg per day, 100ng-5μg per day, 500ng-5μg per day, 1μg-5μg per day, 0.1ng-1μg per day, 0.5ng-1μg per day, 1ng-1μg per day, 5ng-1μg per day, 10ng-1μg per day, 50ng-1μg per day, 100ng-1μg per day, 500 ng-1μg, 0.1ng-500ng per day, 0.5ng-500ng per day, 1ng-500ng per day, 5ng-500ng per day, 10ng-500ng per day, 50ng-500ng per day, 100ng-500ng per day, 0.1ng-100ng per day, 0.5ng-100ng per day, 1ng-100ng per day, 5ng-100ng per day, 10ng-100ng per day, 50ng-100ng per day, 0.1ng-50ng per day, 0.It is consumed in the following ratios: 5ng-50ng, 1ng-50ng per day, 5ng-50ng per day, 10ng-50ng per day, 0.1ng-10ng per day, 0.5ng-10ng per day, 1ng-10ng per day, 5ng-10ng per day, 0.1ng-5ng per day, 0.5ng-5ng per day, 1ng-5ng per day, 0.1ng-1ng per day, 0.1ng-1ng per day, or 0.1ng-0.5ng per day.

[0131] In some embodiments, the microbial compositions of the invention are administered for a period of at least 1 day to 1 week, 1 week to 1 month, 1 month to 3 months, 3 months to 6 months, 6 months to 1 year, or greater than 1 year. For example, in some embodiments, the microbial compositions of the invention are administered for a period of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.

[0132] In some embodiments, the microbial community of the present invention, for example, a high complexity defined gut microbial community, is administered as a single dose or multiple doses. For example, in some embodiments, the microbial community of the present invention, for example, a high complexity defined gut microbial community, is administered once a day, for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the microbial community of the present invention, for example, a high complexity defined gut microbial community, is administered multiple times daily. For example, in some embodiments, the microbial community of the present invention, for example, a high complexity defined gut microbial community, is administered twice a day, three times a day, four times a day, or five times a day. In some embodiments, the microbial community of the present invention, for example, a high complexity defined gut microbial community, is administered intermittently. For example, in some embodiments, the microbial community of the invention, e.g., a high complexity defined gut microbial community, is administered once weekly, once monthly, or when the subject needs it. 12. Combination Therapy

[0133] In some embodiments, the microbial community of the present invention, for example, a high-complexity defined gut microbial community, can be administered in combination with other agents.For example, in some embodiments, the microbial community of the present invention, for example, a high-complexity defined gut microbial community, can be administered in conjunction with or after an antibacterial agent, an antifungal agent, an antiviral agent, an antiparasitic agent, or a prebiotic.Administration can be sequential over a period of hours or days, or simultaneous.

[0134] For example, in some embodiments, the microbial community, e.g., a defined gut microbial community of high complexity, is selected from the group consisting of fluoroquinolone antibiotics (e.g., ciprofloxacin, levaquin, floxin, tequin, avelox, and norflox), cephalosporin antibiotics (e.g., cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprozil). The antibacterial agent may be administered in conjunction with or subsequent to one or more antibacterial agents selected from: anti-inflammatory drugs (e.g., amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); penicillin antibiotics (e.g., amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); tetracycline antibiotics (e.g., tetracycline, minocycline, oxytetracycline, and doxycycline); and carbapenem antibiotics (e.g., ertapenem, doripenem, imipenem / cilastatin, and meropenem).

[0135] For example, in some embodiments, the microbial community, e.g., a defined gut microbial community of high complexity, is selected from the group consisting of abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuviltide, etravirine, famciclovir, foscamet, fomivirsen, ganciclovir, indinavir, idoxuridine, lamivudine, ropinavir, serotonin ... The drug may be administered in conjunction with or subsequent to one or more antiviral agents selected from: valproate, maraviroc, MK-2048, nelfinavir, nevirapine, penciclovir, raltegravir, rilpivirine, ritonavir, saquinavir, stavudine, tenofovir, trifluridine, valacyclovir, valganciclovir, vidarabine, ibasitabine, amantadine, oseltamivir, rimantidine, tipranavir, zalcitabine, zanamivir, and zidovudine.

[0136] In some embodiments, the microbial community, e.g., a defined gut microbial community of high complexity, is selected from the group consisting of miconazole, ketoconazole, clotrimazole, econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole; triazole antifungals, e.g., fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and albaconazole; thiazoline; The antifungal agent may be administered in conjunction with or subsequent to one or more antifungal agents selected from: allylamine antifungals, such as abafungin; allylamine antifungals, such as terbinafine, naftifine, and butenafine; and echinocandin antifungals, such as anidulafungin, caspofungin, and micafungin; poligodial; benzoic acid; ciclopirox; tolnaftate; undecylenic acid; flucytosine or 5-fluorocytosine; griseofulvin; and haloprogin.

[0137] In some embodiments, the microbial community, e.g., a high complexity defined gut microbial community, can be administered in conjunction with or following one or more anti-inflammatory and / or immunosuppressive agents selected from corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, cyclosporine A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anticholinergics, monoclonal anti-IgE antibodies, and vaccines.

[0138] In some embodiments, the microbial communities of the present invention, e.g., high complexity defined gut microbial communities, can be administered in conjunction with or following one or more prebiotics selected from, but not limited to, amino acids, biotin, fructooligosaccharides, galactooligosaccharides, inulin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose, trans-galactooligosaccharides, and xylooligosaccharides. EXAMPLES

[0139] The disclosure, generally described herein, will be more readily understood by reference to the following examples, which are included solely for purposes of illustration of certain aspects and embodiments of the disclosure and are not intended to limit the scope of the disclosure in any way. Example 1 Preparation and optimization of a high-complexity defined gut microbial community

[0140] Figure 1 shows a workflow schematic for the preparation and optimization of a high-complexity defined gut microbial community. Defined microbial strains purchased from the American Type Culture Collection (ATCC, Manassas, VA) were assembled as frozen glycerol stock collections in 96-well plate format. Defined microbial strains were incubated in aliquots in growth medium in 96-well plate format according to the supplier's instructions ("Working Defined Microbial Strain Collection"). The defined microbial strains were recovered by culturing them under culture conditions according to the OD 2010 ... Strains identified as having "jumped in" to the community were identified (by metagenomic analysis), harvested, cultured, and, if necessary, added to the high-complexity defined gut microbial community to generate a new high-complexity defined gut microbial community. Conversely, strains identified as having "dropped out" from the community (by metagenomic analysis) were excluded from the new high-complexity defined gut microbial community. DNA extraction

[0141] DNA was extracted from stool samples using a Qiagen DNesay Power Soil Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Alternative methods for extracting DNA from stool samples are well known and routinely practiced in the art (e.g., as described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3d ed., 2001). Metagenomic analysis

[0142] Sequencing of DNA samples was performed using the TruSeq Nano DNA Library Preparation kit (Illumina, San Diego, CA, US) and the NextSeq platform (Illumina, San Diego, CA, US). Briefly, sequencing libraries were prepared from DNA extracted from each sample. DNA was mechanically fragmented using an ultrasonograph. The fragmented DNA was subjected to end repair and size selection of fragments, adenylation of the 3' ends, ligated with adapters, and enriched for DNA fragments according to the TruSeq Nano DNA Library Preparation kit manual (Illumina, San Diego, CA, US). Samples were sequenced to generate 30–40 million pairs of end read data with a length of 75 bp.

[0143] Each metagenomic analysis was performed with the Metagenomic Intra-Species Diversity Analysis System (MIDAS) (see Nayfach et al., 2016, “An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial transmission and biogeography,” Genome Res. 26 (11): 1612-1625), which estimates the sequencing depth and relative abundance of each microbial species in the stool samples by mapping reads to a reference database of 15 gene families of 5,952 bacterial species, each occurring at one copy per genome in nearly all bacterial genomes. Backfill

[0144] Defined microbial strains that did not colonize (i.e., dropped out) in the microbial community were identified by metagenomic analysis as described above. Similarly, human fecal microbe-exposed microbial strains that colonized (i.e., jumped in) in the mouse intestine were identified by metagenomic analysis as described above. After the first human fecal microbe exposure, of the 104 defined microbial strains inoculated, 97 defined microbial strains persisted in the fecal samples of the exposed mice, and 7 defined microbial strains dropped out. In two mice, 26 human fecal microbe-exposed microbial strains jumped in, and in one mouse, 44 human fecal microbe-exposed microbial strains jumped in. Twenty-two of the 26 microbial strains that jumped into the microbial community in the two exposed mice were obtained from ATCC, generating a high-complexity defined gut microbial community consisting of 119 defined microbial strains in addition to the 97 defined microbial strains that persisted after human fecal microbial exposure (see Table 3 and Figure 2 , “invader strains” = microbial strains that “dropped in” into the community; “input” = defined microbial strains inoculated into the mice). Table 3 [Table 3] Example 2 Treatment of mice with persistent C. difficile infection

[0145] Gnotobiotic 6-8 week old, female Swiss Webster mice were colonized by oral gavage with human fecal samples (200 μl human feces diluted in an equal volume of PBS). Fecal samples from colonized mice were collected weekly for 4 consecutive weeks and frozen for subsequent DNA extraction and metagenomic analysis (as described in Example 1). Four weeks after human stool colonization, mice were treated by oral gavage with 200 μl of 1 mg / ml clindamycin. 24 hours after clindamycin treatment, mice were orally gavaged with 200 μl of a turbid overnight culture of C. difficile and maintained on a high carbohydrate diet. Fecal samples from inoculated mice were collected daily for 3 days after inoculation for CFU plating and frozen for subsequent DNA extraction and metagenomic analysis. Three days after inoculation with C. difficile, mice were treated with human fecal samples, a high-complexity defined gut microbial community of 119 strains, or a phosphate-buffered saline (PBS) vehicle control. Fecal samples from treated mice were collected daily for four days for CFU plating and frozen for subsequent DNA extraction and metagenomic analysis. Four days after treatment, mice were sacrificed and colon samples (e.g., cecum) were prepared for mass spectrometry and histological analysis. See Figure 3A for a schematic workflow of C. difficile infection and treatment schedule. CFU plating

[0146] The fecal samples were diluted with PBS, homogenized using a vortex mixer, and pelleted. The supernatant was used to extract 1 × 10 -1 ~10 -5 Ten-fold serial dilutions of were made in PBS. 100 μl aliquots of each dilution were plated onto CDDC selective agar (see Table 4). [Table 4]

[0147] After 48 hours of anaerobic incubation at 37°C, plates were examined for the growth of colonies with morphology characteristic of C. difficile. Plates with 30-300 colonies were counted using a detection limit of 3.0 log 10 CFU / g were counted. For each dilution, the average of two replicate plates was calculated. If two serial dilutions produced between 30 and 300 colonies, the average count of both dilutions was calculated.

[0148] As shown in Figure 3B, mice receiving treatment with human fecal samples or a high-complexity defined gut microbial community of 119 defined microbial strains had significantly reduced C. difficile CFU / μl counts in fecal samples collected 6 days after C. difficile infection (i.e., 3 days after treatment) compared to mice treated with PBS alone. Example 3 Bile acid analysis by mass spectrometry

[0149] Frozen fecal samples or homogenized cecal sections were pelleted in centrifuge tubes and extracted with ethyl acetate, which was evaporated under vacuum and the pellet redissolved in 200 μl of 20% DMSO / MeOH.

[0150] LC-MS / MS was performed on an Agilent 6120 quadrupole mass spectrometer in negative mode using a Kinetex C18 stationary phase (1.7 μm) column.

[0151] As shown in Figure 4, bile acid concentrations in fecal samples (Figure 4A) and cecal homogenates (Figure 4B) collected from mice treated with human fecal samples and mice treated with a high-complexity defined gut microbial community of 119 defined microbial strains had similar bile acid profiles and concentrations as quantified by MS. Example 4 Metabolite analysis by mass spectrometry

[0152] Urine samples were thawed at room temperature and centrifuged at 13,000×g for 15 min at 4° C. to remove particulate matter. Two volumes of ethyl acetate per volume of urine sample were added and the solution was vortex mixed to precipitate proteins. Ethyl acetate was removed by rotary evaporation. The dried material was dissolved in 80% MeOH / DMSO and separated by reversed-phase HPLC (Agilent 1200 series) for small molecule purification. NMR spectra were collected on either a Bruker Avance DRX500 or Bruker AvanceIII 600-I spectrometer. Purification of the ethyl acetate fraction was performed by gradient HPLC on a C18 reversed-phase column.

[0153] As shown in Figure 5, urine samples collected from mice treated with human fecal samples and mice treated with a high-complexity defined gut microbial community of 119 defined microbial strains had similar bile acid profiles and concentrations as quantified by MS. Example 5 Molecular identification of microbial species Whole-genome shotgun sequencing

[0154] DNA extraction and whole genome shotgun sequencing from isolated microbial cultures or stool samples is performed as previously described in Example 1. Sequence read data are mapped against a comprehensive database of complete sequenced genomes of all defined microbial strains that constitute the gut community. 16S rRNA sequencing

[0155] Molecular identification by 16S rRNA sequencing of microbial colonies in liquid culture or resuspended in PBS is performed by methods known to those skilled in the art (e.g., Turner et al. (see Shin et al., 1999, "Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis," J Eukaryot Microbiol. 46:327-338; Shin et al., 2016, "Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing," Sci Rep. 6:29681). For each defined microbial strain, obtain at least 1300 bp of 16S rRNA sequence for species-level identification. MALDI-TOF MS

[0156] Molecular identification of microbial colonies in liquid culture or resuspended in PBS by MALDI-TOF MS is performed by methods known to those skilled in the art (see, for example, Seuylemezian et al., 2018, “Development of a Custom MALDI-TOF MS Database for Species-Level Identification of Bacterial Isolates Collected From Spacecraft and Associated (See, “Surfaces,” Front Micrbiol. 9:780). Briefly, spots of microbial isolates are transferred to wells of 48- or 96-well plates, layered with 1 μl of 70% formic acid, and air-dried. 1 μl of α-cyano-4-hydroxycinnamic acid matrix in 50% acetonitrile-25% trifluoroacetic acid is layered onto the samples and air-dried. MALDI-TOF MS is performed, for example, using a microbflex LT benchtop mass spectrometer (Bruker Daltonics, Billerica, MA). Spectral data processing is performed, for example, using flexAnalysis software (Bruker Daltonics, Billerica, MA). For each isolate, at least 10 spectra are calculated to generate a major spectral profile, where each spectral line constituting the major spectral profile has a log score higher than 2.7 and a peak number higher than 75%. It has frequency OLE_LINK1. Example 6 Treatment methods for persistent C. difficile infections

[0157] The high complexity defined gut microbial community of the present invention is administered in an effective amount for the treatment of persistent C. difficile infection in a mammalian subject in need thereof. The high complexity defined gut microbial community is administered as a composition formulated for oral administration or other non-parenteral route of administration as described herein. The mammalian subject may or may not have been treated with an antibiotic prior to treatment with the high complexity defined gut microbial community. After one treatment, the mammalian subject has improved symptoms associated with persistent C. difficile infection or has a significant reduction in the number of C. difficile CFUs in the intestine of the mammalian subject. Alternatively, after two or more treatments, the mammalian subject has improved symptoms associated with persistent C. difficile infection or has a significant reduction in the number of C. difficile CFUs in the intestine of the mammalian subject. Example 7 Treatment methods for cholestatic diseases

[0158] The high complexity defined gut microbial community of the present invention is administered in an effective amount for treating cholestatic disease in a mammalian subject in need thereof. The high complexity defined gut microbial community is administered as a composition formulated for oral administration or other non-parenteral routes of administration as described herein. The mammalian subject may or may not have been treated with antibiotics prior to treatment with the high complexity defined gut microbial community. After one treatment, the mammalian subject is improved in symptoms associated with cholestatic disease or the bile acid composition profile and / or concentration in the intestine of the mammalian subject is significantly modified. Alternatively, after two or more treatments, the mammalian subject is improved in symptoms associated with cholestatic disease or the bile acid composition profile and / or concentration in the intestine of the mammalian subject is significantly modified. Incorporation by Reference

[0159] The entire disclosures of each of the patent documents and scientific articles referenced herein are incorporated by reference for all purposes. Equivalent

[0160] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the above specification, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. The present invention provides, for example, the following items. (Item 1) A high-complexity defined gut microbial community comprising a plurality of between 40 and 500 defined microbial strains, said defined gut microbial community achieves substantial engraftment when administered to gnotobiotic mice; A high complexity defined gut microbial community, wherein the engrafted defined gut microbial community is stable following exposure to human fecal community microbes. (Item 2) 2. The high complexity defined gut microbial community according to item 1, wherein community stability is characterized by up to 10% of the defined microbial strains dropping out after exposure to the microorganisms. (Item 3) 3. The defined gut microbial community of high complexity according to item 1 or 2, wherein the community stability is characterized by a maximum of 10% emergence of new strains resulting from the human fecal community appearing after exposure to the microorganisms. (Item 4) 2. The high complexity defined gut microbial community according to item 1, wherein at least 50% of the defined microbial strains are detectable after exposure to the microbial organism. (Item 5) 5. The high complexity defined gut microbial community according to item 4, wherein at least 60% of the defined microbial strains are detectable after exposure to the microbial organism. (Item 6) 6. The high complexity defined gut microbial community according to item 5, wherein at least 70% of the defined microbial strains are detectable after exposure to the microbial organism. (Item 7) 7. The high complexity defined gut microbial community according to item 6, wherein at least 80% of the defined microbial strains are detectable after exposure to the microorganism. (Item 8) 8. The high complexity defined gut microbial community according to item 7, wherein at least 90% of the defined microbial strains are detectable after exposure to the microbial organism. (Item 9) 9. The high complexity defined gut microbial community according to item 8, wherein at least 95% of the defined microbial strains are detectable after exposure to the microorganism. (Item 10) 10. The high complexity defined gut microbial community according to item 9, wherein at least 99% of the defined microbial strains are detectable after exposure to the microbial organism. (Item 11) 11. The high complexity defined gut microbial community of any one of items 1 to 10, wherein community stability is characterized by metagenomic analysis of fecal samples obtained from said mice after microbial exposure. (Item 12) Item 13. The high-complexity defined gut microbial community according to item 11, wherein the metagenomic analysis is selected from whole genome sequencing, ribosomal gene sequencing, or ribosomal RNA sequencing. 13. The high complexity defined gut microbial community according to item 12, wherein the whole genome sequencing is whole genome shotgun sequencing. (Item 14) 14. The high complexity defined gut microbial community according to any one of items 1 to 13, wherein the defined gut microbial community comprises between 100 and 200 defined microbial strains. (Item 15) 15. The high complexity defined gut microbial community according to item 14, wherein the defined gut microbial community comprises between 100 and 130 defined microbial strains. (Item 16) 16. The high complexity defined gut microbial community according to any one of items 1 to 15, wherein each defined microbial strain is molecularly identified. (Item 17) 17. The high complexity defined gut microbial community of item 16, wherein the molecular identification comprises the identification of a nucleic acid sequence that uniquely identifies each of the defined microbial strains. (Item 18) 18. The high complexity defined gut microbial community according to item 17, wherein the nucleic acid sequence comprises a 16S rRNA sequence. (Item 19) 18. The high complexity defined gut microbial community of item 17, wherein the nucleic acid sequence comprises a whole genome sequence. (Item 20) 17. The high complexity defined gut microbial community according to item 16, wherein the molecular identification comprises matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. (Item 21) 20. The high complexity defined gut microbial community according to any one of items 1 to 19, wherein when tested in a mouse model of persistent Clostridium difficile infection, the defined gut microbial community reduces the number of colony forming units (cfu) of C. difficile per μl of faeces by at least 1-2 log. (Item 22) 22. The high complexity defined gut microbial community according to item 21, wherein when tested in a mouse model of persistent C. difficile infection, the defined gut microbial community reduces the number of C. difficile cfu per μl of feces by at least 2-3 logs. (Item 23) 23. The high complexity defined gut microbial community according to item 22, wherein the defined gut microbial community reduces the number of C. difficile cfu per μl of feces by at least 3 to 4 logs when tested in a mouse model of persistent C. difficile infection. (Item 24) 24. The high complexity defined gut microbial community according to item 23, wherein the defined gut microbial community reduces the number of C. difficile cfu per μl of feces by at least 4-5 logs when tested in a mouse model of persistent C. difficile infection. (Item 25) 25. The high complexity defined gut microbial community according to item 24, wherein when tested in a mouse model of persistent C. difficile infection, the defined gut microbial community reduces the number of C. difficile cfu per μl of feces by at least 5-6 logs. (Item 26) 26. The high complexity defined gut microbial community of any one of items 1 to 25, wherein said defined gut microbial community significantly alters the profile of bile acids present in the faeces of said mice compared to isogenic gnotobiotic control mice. (Item 27) 27. The high complexity defined gut microbial community of any one of items 1 to 26, wherein the defined gut microbial community significantly alters the concentration of bile acids present in the faeces of the mouse compared to an isogenic gnotobiotic control mouse. (Item 28) 28. The high complexity defined gut microbial community according to item 26 or 27, wherein the bile acids are selected from the group consisting of Tβ-MCA, Tα-MCA, TUDCA, THDCA, TCA, 7β-CA, 7-oxo-CA, TCDCA, Tω-MCA, TDCA, α-MCA, β-MCA, ω-MCA, Muro-CA, d4-CA, CA, TLCA, UDCA, HDCA, CDCA, DCA and LCA. (Item 29) 29. The high complexity defined gut microbial community of any one of items 1 to 28, wherein the defined gut microbial community significantly alters the concentration of one or more metabolites in the urine of the mouse compared to an isogenic gnotobiotic control mouse. (Item 30) The one or more metabolites are 4-hydroxybenzoic acid, L-tyrosine, 4-hydroxyphenylacetic acid, DL-p-hydroxyphenyllactic acid, p-coumaric acid, 3-(4-hydroxyphenyl)propionic acid, 3-(4-hydroxyphenyl)pyruvic acid, indole-3-carboxylic acid, tyramine, L-phenylalanine, phenylacetic acid, 3-indole 30. The high complexity defined gut microbial community according to item 29, wherein the microbial components are selected from the group consisting of acetic acid, DL-3-phenyllactic acid, L-tryptophan, DL-indole-3-lactic acid, phenylpyruvic acid, trans-3-indoleacrylic acid, 3-indolepyruvic acid, 3-indolepropionic acid, 3-phenylpropionic acid, trans-cinnamic acid, tryptamine, phenol, indole-3-carboxaldehyde, p-cresol, indole, 4-vinylphenol, and 4-ethylphenol. (Item 31) 31. The high complexity defined gut microbial community according to any one of items 1 to 30, wherein one or more of said defined microbial strains have at least two metabolic phenotypes selected from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. (Item 32) 32. The high complexity defined gut microbial community according to item 31, wherein one or more of the defined microbial strains have at least three metabolic phenotypes selected from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. (Item 33) 33. The high complexity defined gut microbial community according to item 32, wherein one or more of the defined microbial strains have at least five metabolic phenotypes selected from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. (Item 34) 34. The high complexity defined gut microbial community according to item 33, wherein one or more of the defined microbial strains have at least 10 metabolic phenotypes selected from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. (Item 35) 35. The high complexity defined gut microbial community according to item 34, wherein one or more of the defined microbial strains have all metabolic phenotypes from the group consisting of mucin degradation, polysaccharide fermentation, hydrogen utilization, succinate metabolism, butyrate production, amino acid metabolism, bile acid metabolism, CO2 fixation, formate metabolism, methanogenesis, acetate production, hydrogen production, and propionate production. (Item 36) The microbial strain defined above is Acidaminococcus fermentans DSM 20731、Acidaminococcus sp.D21、Akkermansia muciniphila ATCC BAA-835、Alistipes putredinis DSM 17216、Anaerofustis stercorihominis DSM 17244、Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroide s sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium adolescentis L2-32、Bifidobacterium breve DSM 20213、Bifidobacterium catenulatum DSM 16992、Bifidobacterium longum infantis ATCC 55813、Bifidobacterium pseudocatenulatum DSM 20438、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Bryantella formatexigens DSM 14469、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathewayi DSM 13479、Clostridium hylemonas DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.L2-50、Clostridium sp.M62 / 1、Clostridium spiroforme DSM 1552、Clostridium sporogenes ATCC 15579、Collinsella aerofaciens ATCC 25986、Collinsella stercoris DSM 13279、Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dialister invisus DSM 15470, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Lactobacillus ruminis ATCC 25644, Lactococcus lactis subsp.lactis Il1403 -> sub DSMZ 20729, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544、Olsenella uli DSM 7084、Parabacteroides distasonis ATCC 8503、Parabacte A defined gut microbiota of high complexity according to any one of items 1 to 35, comprising roides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp. D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii L2-32, Ruminococcus flavefaciens FD 1, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum variabile DSM 15176, Veillonella dispar ATCC 17748, Veillonella sp. 3_1_44 HM 64, and Veillonella sp. 6_1_27 HM 49. (Item 37) The defined microbial strains are Acidaminococcus fermentans DSM 20731, Acidaminococcus sp. D21, Akkermansia muciniphila ATCC BAA-835, Alistipes putredinis DSM 17216, Anaerofustis stercorihominis DSM 17244, Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium adolescentis L2-32、Bifidobacterium breve DSM 20213、Bifidobacterium catenulatum DSM 16992、Bifidobacterium longum infantis ATCC 55813、Bifidoba cterium pseudocatenulatum DSM 20438、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Bryantella formatexigens DSM 14469、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathewayi DSM 13479、Clostridium hylemonas DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.L2-50、Clostridium sp.M62 / 1、Clostridium spiroforme DSM 1552、Clostridium sporogenes ATCC 15579、Collinsella aerofaciens ATCC 25986、Collinsella stercoris DSM 13279、Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dialister invisus DSM 15470, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Lactobacillus ruminis ATCC 25644, Lactococcus lactis subsp.lactis Il1403 -> sub DSMZ 20729, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Olsenella uli DSM 7084, Parabacteroides distasonis ATCC 8503, Parabacteroides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp. D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii L2-32, Ruminococcus flavefaciens FD 1, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 The defined intestinal microbiota of high complexity according to item 36, consisting of DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum variabile DSM 15176, Veillonella dispar ATCC 17748, Veillonella sp. 3_1_44 HM 64, and Veillonella sp. 6_1_27 HM 49. (Item 38) The defined microbial strains are Acidaminococcus fermentans DSM 20731, Acidaminococcus sp.D21, Adlercreutzia equolifaciens DSM 19450, Akkermansia muciniphila ATCC BAA-835, Alistipes finegoldii DSM 17242, Alistipes ihumii AP11, Alistipes indistinctus YIT 12060 / DSM 22520, Alistipes onderdonkii DSM 19147, Alistipes putredinis DSM 17216, Alistipes senegalensis JC50 / DSM 25460, Alistipes shahii WAL 8301 / DSM 19121, Anaerofustis stercorihominis DSM 17244, Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides rodentium DSM 26882、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis、ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium breve、Bifidobacterium catenulatum DSM 16992、Bifidobacterium pseudocatenulatum DSM 20438、Bilophila wadsworthia ATCC 49260、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Blautia sp.KLE 1732(HM 1032)、Blautia wexlerae DSM 19850、Bryantella formatexigens DSM 14469、Burkholderiales bacterium 1_1_47、Butyricimonas virosa DSM 23226、Butyrivibrio crossotus DSM 2876、Catenibacterium m itsuokai DSM 15897、Clostridiales bacterium VE202-03、Clostridiales bacterium VE202-14、Clostridiales bacterium VE202-27、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathewayi DSM 13479、Clostridium hylemonae DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.ATCC 29733 VPI C48-50、Clostridium sp.L2-50、Clostridium sp.M62 / 1, Clostridium spiroforme DSM 1552, Collinsella aerofaciens ATCC 25986, Collinsella stercoris DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Intestinimonas butyriciproducens DSM 26588, Lactobacillus ruminis ATCC 25644, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Odoribacter splanchnicus DSM 20712, Olsenella uli DSM 7084, Oscillibacter sp.KLE 1728, Parabacteroides distasonis ATCC 8503, Parabacteroides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp. D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii ATCC, Ruminococcus flavefaciens FD1, Ruminococcus gauvreauii DSM 19829, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrini. The highly complex defined intestinal microbiota according to any one of items 1 to 35, comprising reducens DSM 20476, Solobacterium moorei DSM 22971, Streptococcus thermophilus LMD-9 (ATCC 19258), Subdoligranulum sp. 4_3_54A2FAA, Subdoligranulum variabile DSM 15176, and Veillonella dispar ATCC 17748. (Item 39) The defined microbial strain is Acidaminococcus fermentans DSM 20731, Acidaminococcus sp.D21, Adlercreutzia equolifaciens DSM 19450, Akkermansia muciniphila ATCC BAA-835, Alistipes finegoldii DSM 17242, Alistipes ihumii AP11, Alistipes indistinctus YIT 12060 / DSM 22520, Alistipes onderdonkii DSM 19147, Alistipes putredinis DSM 17216, Alistipes senegalensis JC50 / DSM 25460, Alistipes shahii WAL 8301 / DSM 19121, Anaerofustis stercorihominis DSM 17244, Anaerostipes caccae DSM 14662、Anaerotruncus colihominis DSM 17241、Bacteroides caccae ATCC 43185、Bacteroides cellulosilyticus DSM 14838、Bacteroides coprocola DSM 17136、Bacteroides coprophilus DSM 18228、Bacteroides dorei 5_1_36 / D4(HM 29)、Bacteroides dorei DSM 17855、Bacteroides eggerthii DSM 20697、Bacteroides finegoldii DSM 17565、Bacteroides fragilis 3_1_12、Bacteroides intestinalis DSM 17393、Bacteroides ovatus ATCC 8483、Bacteroides pectinophilus ATCC 43243、Bacteroides plebeius DSM 17135、Bacteroides rodentium DSM 26882、Bacteroides sp.1_1_6、Bacteroides sp.2_1_16、Bacteroides sp.2_1_22、Bacteroides sp.3_1_19、Bacteroides sp.9_1_42FAA、Bacteroides sp.D2、Bacteroides stercoris ATCC 43183 DSMZ 19555、Bacteroides thetaiotaomicron VPI-5482、Bacteroides uniformis、ATCC 8492、Bacteroides vulgatus ATCC 8482、Bacteroides xylanisolvens SD CC 1b -> subbed w / DSMZ 18836、Bifidobacterium breve、Bifidobacterium catenulatum DSM 16992、Bifidobacterium pseudocatenulatum DSM 20438、Bilophila wadsworthia ATCC 49260、Blautia hansenii DSM 20583、Blautia hydrogenotrophica DSM 10507、Blautia sp.KLE 1732(HM 1032)、Blautia wexlerae DSM 19850、Bryantella formatexigens DSM 14469、Burkholderiales bacterium 1_1_47、Butyricimonas virosa DSM 23226、Butyrivibrio crossotus DSM 2876、Catenibacterium mitsuokai DSM 15897、Clostridiales bacteri um VE202-03、Clostridiales bacterium VE202-14、Clostridiales bacterium VE202-27、Clostridium asparagiforme DSM 15981、Clostridium bartlettii DSM 16795、Clostridium bolteae ATCC BAA-613、Clostridium hathewayi DSM 13479、Clostridium hylemonae DSM 15053、Clostridium leptum DSM 753、Clostridium methylpentosum DSM 5476、Clostridium nexile DSM 1787、Clostridium saccharolyticum WM1 DSMZ 2544、Clostridium scindens ATCC 35704、Clostridium sp.ATCC 29733 VPI C48-50、Clostridium sp.L2-50、Clostridium sp.M62 / 1, Clostridium spiroforme DSM 1552, Collinsella aerofaciens ATCC 25986, Collinsella stercoris DSM 13279, Coprococcus comes ATCC 27758, Coprococcus eutactus ATCC 27759, Desulfovibrio piger ATCC 29098, Dorea formicigenerans ATCC 27755, Dorea longicatena DSM 13814, Eggerthella lenta DSM 2243, Ethanoligenens harbinense YUAN-3 DSMZ 18485, Eubacterium biforme DSM 3989, Eubacterium dolichum DSM 3991, Eubacterium eligens ATCC 27750 DSMZ 3376, Eubacterium hallii DSM 3353, Eubacterium rectale ATCC 33656, Eubacterium siraeum DSM 15702, Eubacterium ventriosum ATCC 27560 DSM 3988, Faecalibacterium prausnitzii A2-165, Granulicatella adiacens ATCC 49175 DSMZ 9848, Holdemania filiformis DSM 12042, Intestinimonas butyriciproducens DSM 26588, Lactobacillus ruminis ATCC 25644, Megasphaera DSMZ 102144, Mitsuokella multacida DSM 20544, Odoribacter splanchnicus DSM 20712, Olsenella uli DSM 7084, Oscillibacter sp.KLE 1728, Parabacteroides distasonis ATCC 8503, Parabacteroides johnsonii DSM 18315, Parabacteroides merdae ATCC 43184 DSMZ 19495, Parabacteroides sp.D13, Prevotella buccae D17, Prevotella buccalis ATCC 35310 DSMZ 20616, Prevotella copri DSM 18205, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Ruminococcus albus strain 8, Ruminococcus bromii ATCC, Ruminococcus flavefaciens FD 1, Ruminococcus gauvreauii DSM 19829, Ruminococcus gnavus ATCC 29149, Ruminococcus lactaris ATCC 29176, Ruminococcus obeum ATCC 29174, Ruminococcus torques ATCC 27756, Slackia exigua ATCC 700122 DSMZ 15923, Slackia heliotrinireducens DSM 20476, Solobacterium moorei. A defined gut microbial community of high complexity as described in item 38 consisting of Subdoligranulum sp. 4_3_54A2FAA, Subdoligranulum variabile DSM 15176, and Veillonella dispar ATCC 17748. (Item 40) 40. A method for treating an animal having a microbial incompatibility or pathology comprising administering a high complexity defined gut microbial community according to any one of items 1 to 39. (Item 41) 41. The method of claim 40, wherein the animal is a mammal. (Item 42) 42. The method of claim 41, wherein the animal is a human. (Item 43) 43. The method of any one of items 40 to 42, wherein the dysbiosis or pathology is a persistent C. difficile infection. (Item 44) 43. The method according to any one of items 40 to 42, wherein the dysbiosis or condition is a cholestatic disease. (Item 45) 45. The method of claim 44, wherein the cholestatic disease is selected from the group consisting of primary sclerosing cholangitis, primary biliary cholangitis, progressive familial intrahepatic cholestasis, and nonalcoholic steatohepatitis. (Item 46) 46. ​​The method of any one of items 40 to 45, wherein said high complexity defined gut microbial community is administered via a route selected from the group consisting of oral, rectal, fecal (by enema), and naso / oral gavage. (Item 47) 40. The method of any one of items 1 to 39 for generating a high complexity defined gut microbial community, wherein each of the plurality of defined microbial strains is cultured individually and then combined to form the defined gut microbial community. (Item 48) 40. The method of any one of items 1 to 39, wherein all of said plurality of defined microbial strains are cultured together to form said defined gut microbial community. (Item 49) 40. The method of generating a high complexity defined gut microbial community according to any one of the preceding claims, comprising culturing one or more of said plurality of defined microbial strains individually, culturing two or more of said defined microbial strains together, and combining said individually cultured defined microbial strains and said co-cultured defined microbial strains together to form said defined gut microbial community. (Item 50) 40. A formulation comprising a high complexity defined gut microbial community according to any one of items 1 to 39 and a pharma- ceutically acceptable carrier or excipient. (Item 51) 1. A method for generating a high complexity defined gut microbial community by in vivo backfilling, the method comprising: i) combining a plurality of defined microbial strains; ii) engrafting the combined multiple defined microbial strains in the intestine of an animal to generate an engrafted animal; iii) exposing said engrafted animals to a human stool sample; iv) maintaining the exposed engrafted animals for a time sufficient for intestinal colonization of the animals with microbial strains from the human stool sample, thereby generating an intestinal community in the intestine of the animals; v) identifying microbial strains of the gut community by metagenomic analysis; vi) identifying whether there are differences between the microbial strains that make up the gut community and the microbial strains that make up the combined plurality of defined microbial strains; vii) if there is a significant difference between the microbial strains constituting the gut community and the microbial strains constituting the combined plurality of defined microbial strains, adding one or more additional defined microbial strains that were not present in step i) to the combined plurality of defined microbial strains or removing defined microbial strains that were present in the combined plurality of defined microbial strains of step i) to generate a modified combined plurality of defined microbial strains, and repeating steps ii) to vi) in non-engrafted animals using the modified combined plurality of defined microbial strains as the combined plurality of defined microbial strains. and if minimal differences exist, the modified defined microbial community of final step vii) is a high complexity defined gut microbial community. (Item 52) Step i) is selected from the group consisting of fructan, inulin, glucuronoxylan, arabinoxylan, glucomannan, β-mannan, dextran, starch, arabinan, xyloglucan, galacturonan, β-glucan, galactomannan, rhamnogalacturonan I, rhamnogalacturonan II, arabinogalactan, mucin O-linked glycan, yeast α-mannan, yeast β-glucan, chitin, alginic acid, porphyrin, laminarin, carrageenan, agarose, alternan, levan, xanthan gum, galactooligosaccharides, hyaluronan, chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, phenylalanine, tyrosine, tryptophan, leucine, valine, isoleucine, glycine, proline, asparagine, glutamine, and the like. 52. The method of claim 51, comprising combining one or more defined microbial strains capable of converting a substrate selected from the group consisting of: aspartic acid, glutamic acid, cysteine, lysine, arginine, serine, methionine, alanine, arginine, histidine, ornithine, citrulline, carnitine, hydroxyproline, cholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholesterol, cinnamic acid, coumaric acid, sinapic acid, ferulic acid, caffeic acid, quinic acid, chlorogenic acid, catechin, epicatechin, gallic acid, pyrogallol, catechol, quercetin, myricetin, campherol, luteolin, apigenin, naringenin, and hesperidin. (Item 53) 53. The method of claim 52, wherein the combined plurality of defined microbial strains is capable of metabolizing at least 60 of the substrates listed in claim 52. (Item 54) Item 54. The method of item 53, wherein the combined plurality of defined microbial strains is capable of metabolizing at least 70 of the substrates listed in item 52. (Item 55) 55. The method of claim 54, wherein the combined plurality of defined microbial strains is capable of metabolizing at least 80 of the substrates listed in item 52. (Item 56) Item 56. The method of item 55, wherein the combined plurality of defined microbial strains is capable of metabolizing all of the substrates listed in item 52. (Item 57) viii) performing a C. difficile plate count on fecal samples obtained from animals with persistent C. difficile infection; ix) determining whether the high complexity defined gut microbial community is a member of the persistent C. difficile infection group; engrafting said animal with the infection in the intestine to produce an engrafted infected animal; x) maintaining said live-infected animal for a time sufficient for intestinal colonization with microbial strains of said high complexity defined gut microbial community, thereby generating a live-infected community in the intestine of said live-infected animal; xi) performing additional C. difficile plate counts on fecal samples obtained from the engrafted infected animals; xii) if the number of C. difficile CFUs obtained from the plate count in step xi) is not significantly less than the number of C. difficile CFUs obtained from the plate count in step viii), adding one or more additional defined microbial strains that were not present in step ix) to the high complexity defined gut microbial community to generate a modified high complexity defined gut microbial community, and repeating steps viii)-xi) in animals with non-engrafted, persistent C. difficile infection using the modified high complexity defined gut microbial community as the high complexity defined gut microbial community. wherein if there is a statistically significant reduction in the CFU count of C. difficile obtained from the plate count of step xi) compared to the CFU count of C. difficile obtained from the plate count of step viii), the modified high complexity defined gut microbial community of final step xi) is a final high complexity defined gut microbial community.

Claims

[Claim 1] The invention described in this specification.

Images